Polypeptide inhibitors of HSP27 kinase and uses therefor

ABSTRACT

The present invention provides polypeptide inhibitors of HSP27 kinase, compositions thereof, and methods for using such polypeptides and compositions for various therapeutic uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/880,137 filed Jan. 10, 2007 which is incorporated byreference herein in its entirety.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with government support under Grant No. K25HL074968 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 15, 2012, isnamed 32202139.txt and is 26,004 bytes in size.

FIELD OF THE INVENTION

The invention is in the fields of cell and molecular biology,polypeptides, drug discovery, and therapeutic methods of use.

SUMMARY OF THE INVENTION

The present invention provides polypeptide inhibitors of HSP27 kinase,compositions thereof, and methods for using such polypeptides andcompositions for various therapeutic uses.

In one embodiment, the present invention provides a polypeptidecomprising or consisting of a sequence according to general formula I:Z1-X1-X2-X3-X4X5-X6-X7-X8-X9-X10-Z2 (SEQ ID NO: 69)

wherein Z1 and Z2 are independently absent or are transduction domains;

X1 is selected from the group consisting of A, KA, KKA, KKKA (SEQ ID NO:70), and RA, or is absent;

X2 is selected from the group consisting of G, L, A, V, I, M, Y, W, andF, or is an aliphatic amino acid;

X3 is selected from the group consisting of V, L, I, A, G, Q, N, S, T,and C, or is an aliphatic amino acid;

X4 is selected from the group consisting of Q, N, H, R and K;

X5 is selected from the group consisting of Q and N;

X6 is selected from the group consisting of C, A, G, L, V, I, M, Y, W,and F or is an aliphatic amino acid;

X7 is selected from the group consisting of S, A, C, T, and G or is analiphatic amino acid;

X8 is selected from the group consisting of V, L, I, and M;

X9 is absent or is any amino acid; and

X10 is absent or is any amino acid;

wherein at least one of the following is true:

(a) X3 is N and X7 is not G;

(b) X7 is G and X3 is not N;

(c) X2 is not L;

(d) X4 is not R;

(e) X5 is not Q;

(f) X6 is not L;

(g) X8 is not V;

(h) X10 is absent; or

(i) X9 and X10 are absent.

In another embodiment of the isolated polypeptide according to generalformula I, X4 is R, X5 is Q and X8 is V (SEQ ID NO: 71). In anotherembodiment, the polypeptide comprises KAFAKLAARLYRKALARQLGVAA (SEQ IDNO: 48). In another embodiment, the polypeptide comprisesFAKLAARLYRKALARQLGVAA (SEQ ID NO: 49). In another embodiment, theinvention includes variants of SEQ ID NO: 48 that are at least 90%identical to SEQ ID NO: 48 and inhibit TNF-α excretion. In anotherembodiment, the invention includes variants of SEQ ID NO: 49 that are atleast 90% identical to SEQ ID NO: 49 and inhibit TNF-α excretion.

The present invention further provides compositions comprising one ormore isolated polypeptides comprising a sequence according to generalformula I and a pharmaceutically acceptable carrier: In one embodiment,the composition comprises the isolated polypeptideKAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 48) and a pharmaceuticallyacceptable carrier. In another embodiment, the composition comprises theisolated polypeptide FAKLAARLYRKALARQLGVAA (SEQ ID NO: 49) and apharmaceutically acceptable carrier. In another embodiment, thecomposition comprises variants of SEQ ID NO: 48 that are at least 90%identical to SEQ ID NO: 48 and inhibit TNF-α excretion and apharmaceutically acceptable carrier. In another embodiment, thecomposition comprises variants of SEQ ID NO: 49 that are at least 90%identical to SEQ ID NO: 49 and inhibit TNF-α excretion and apharmaceutically acceptable carrier.

The invention also provides an isolated nucleic acid sequence encodingone or more isolated polypeptides comprising a sequence according togeneral formula I: In one embodiment, the isolated nucleic acid encodesthe isolated polypeptide KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 48). Inanother embodiment, the isolated nucleic acid encodes the isolatedpolypeptide FAKLAARLYRKALARQLGVAA (SEQ ID NO: 49). In anotherembodiment, In one embodiment, the isolated nucleic acid encodesvariants of SEQ ID NO: 48 that are at least 90% identical to SEQ ID NO:48 and inhibit TNF-α excretion. In another embodiment, the In oneembodiment, the isolated nucleic acid encodes variants of SEQ ID NO: 49that are at least 90% identical to SEQ ID NO: 49 and inhibit TNF-αexcretion. The present invention also provides recombination expressionvectors comprising these nucleic acids and a host cell transfected withthe recombinant expression vectors.

The present invention further provides a biomedical device comprisingone or more isolated polypeptides comprising a sequence according togeneral formula 1, wherein the one or more isolated polypeptides aredisposed on or in the device. In one embodiment, the biomedical devicecomprises the polypeptide of SEQ ID NO: 48. In another embodiment, thebiomedical device comprises the polypeptide of SEQ ID NO: 49. In anotherembodiment, the biomedical device comprises variants of SEQ ID NO: 48that are at least 90% identical to SEQ ID NO: 48 and inhibit TNF-αexcretion. In another embodiment, the biomedical device comprisesvariants of SEQ ID NO: 49 that are at least 90% identical to SEQ ID NO:49 and inhibit TNF-α excretion.

The present invention further provides a biomedical device comprisingone or more isolated polypeptides comprising a sequence according togeneral formula 1, where the one or more isolated polypeptides aredisposed in a matrix disposed on the device. In one embodiment, thebiomedical device comprises the polypeptide of SEQ ID NO: 48. In anotherembodiment, the biomedical device comprises the polypeptide of SEQ IDNO: 49. In another embodiment, the biomedical device comprises variantsof SEQ ID NO: 48 that are at least 90% identical to SEQ ID NO: 48 andinhibit TNF-α excretion. In another embodiment, the biomedical devicecomprises variants of SEQ ID NO: 49 that are at least 90% identical toSEQ ID NO: 49 and inhibit TNF-α excretion. In some embodiments, thematrix is a heparin coating.

The present invention moreover provides a method for treating aninflammatory disease, disorder or condition in a subject in needthereof, the method comprising the step of (a) administering atherapeutically effective amount of a composition of the invention. Inone embodiment, the inflammatory disease, disorder, or condition isselected from the group consisting of hyperplastic scarring, keloids,rheumatoid arthritis, chronic obstructive pulmonary disease,atherosclerosis, intimal hyperplasia, Crohn's disease, inflammatorybowel disease, osteoarthritis, Lupus, tendonitis, psoriasis, gliosis,inflammation, type II diabetes mellitus, type I diabetes mellitus,Alzheimer's disease, and adhesions. In another embodiment, the disease,disorder or condition is hyperplastic scarring. In another embodiment,the disease, disorder or condition is rheumatoid arthritis. In anotherembodiment, the disease, disorder or condition is chronic obstructivepulmonary disease. In another embodiment, the disease, disorder orcondition is atherosclerosis. In another embodiment, the disease,disorder or condition is intimal hyperplasia. In another embodiment, thedisease, disorder or condition is Crohn's disease. In anotherembodiment, the disease, disorder or condition is inflammatory boweldisease. In another embodiment, the disease, disorder or condition isosteoarthritis. In another embodiment, the disease, disorder orcondition is tendonitis. In another embodiment, the disease, disorder orcondition is psoriasis. In another embodiment, the disease, disorder orcondition comprises glial scarring. In another embodiment, the disease,disorder or condition is a traumatic brain injury. In anotherembodiment, the disease, disorder or condition is a spinal cord injury.In another embodiment, the method further comprises the steps of (b)monitoring a level of at least one biomarker in a target tissue, whereinthe at least one biomarker is selected from the group consisting of:TGFβ1 expression; collagen I expression; CTGF expression; α-smoothmuscle actin expression; TNF-α; IL-1; IL-6; IL-8; COX-2; MIP-1α; andMIP-2; and (c) maintaining the level of the biomarker in the targettissue substantially at normal levels during treatment.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of the necessary fee.

FIG. 1: Data graph showing that HSP27 kinase inhibitor peptide (BIP)inhibits TGF-β1 induced expression of CTGF (A) and collagen (B) in humankeloid fibroblasts.

FIG. 2: Data graph showing that HSP27 kinase inhibitor peptide (BIP)(SEQ ID NO: 54 ) inhibits TGF-β1 induced stress fiber formation in humankeloid fibroblasts.

FIG. 3: Data graph showing that HSP27 kinase inhibitor peptide (BIP)inhibits TGF-β1 induced phosphorylation of HSP27 in human keloidfibroblasts.

FIG. 4: (A) Immunoblot, and (B) Data graph showing that HSP27 kinaseinhibitor peptide (BIP) inhibits TGF-β1 induced phosphorylation of HSP27in human keloid fibroblasts.

FIG. 5: Data graph showing that HSP27 kinase inhibitor peptide (BIP)(SEQ ID NO: 54) inhibits in vitro phosphorylation of HSP27 by MAPKAPkinase 2. FIG. 5 also discloses “KKKALNRQLGVAA” as SEQ ID NO: 72.

FIG. 6: Data graph showing HSP27 kinase inhibitor peptide (BIP) (SEQ IDNO: 54) enhances sodium nitroprusside-induced relaxation of humansaphenous vein.

FIG. 7: Data graph showing HSP27 kinase inhibitor peptide (BIP) (SEQ IDNO: 54) does not inhibit endothelial cell proliferation.

FIG. 8: Data graph showing cellular viability of LPS-induced THP-1monocytes treated with HSP27 kinase inhibitor peptides KAF (SEQ ID NO:48)and FAK (SEQ ID NO: 49).

FIG. 9: Data graph showing that HSP27 kinase inhibitor peptides KAF (SEQID NO: 48)and FAK (SEQ ID NO: 49) inhibit LPS-induced TNF-α excretion ofTHP-1 monocytes.

FIG. 10: Data graph showing cellular viability of LPS-induced THP-1monocytes treated with HSP27 kinase inhibitor peptides BIP (SEQ ID NO:54) and YARA (“YARA” disclosed as SEQ ID NO: 73, YARA full-lengthpeptide disclosed as SEQ ID NO: 55.

FIG. 11: Data graph showing that HSP27 kinase inhibitor peptides BIP(SEQ ID NO: 54) and YARA (“YARA” disclosed as SEQ ID NO: 73, YARAfull-length peptide disclosed as SEQ ID NO: 55) inhibit LPS-inducedTNF-α excretion of THP-1 monocytes.

FIG. 12: Data graph showing that HSP27 kinase inhibitor peptide KAF (SEQID NO: 48) decreases phosphorylation of HSP27 in astrocytes even in thepresence of TNF-α.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.).

The single letter designation for amino acids is used predominatelyherein. As is well known by one of skill in the art, such single letterdesignations are as follows: A is alanine; C is cysteine; D is asparticacid; E is glutamic acid; F is phenylalanine; G is glycine; H ishistidine; I is isoleucine; K is lysine; L is leucine; M is methionine;N is asparagine; P is proline; Q is glutamine; R is arginine; S isserine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to a “polypeptide” means one or more polypeptides.

In a first aspect, the present invention provides a polypeptidecomprising or consisting of a sequence according to general formula I:

Z1-X1-X2-X3-X4 X5-X6-X7-X8-X9-X10-Z2 (SEQ ID NO: 1)

wherein Z1 and Z2 are independently absent or are transduction domains;

X1 is selected from the group consisting of A, KA, KKA, KKKA (SEQ ID NO:70), and RA, or is absent;

X2 is selected from the group consisting of G, L, A, V, I, M, Y, W, andF, or is an aliphatic amino acid;

X3 is selected from the group consisting of V, L, I, A, G, Q, N, S, T,and C, or is an aliphatic amino acid;

X4 is selected from the group consisting of Q, N, H, R and K;

X5 is selected from the group consisting of Q and N;

X6 is selected from the group consisting of C, A, G, L, V, I, M, Y, W,and F or is an aliphatic amino acid;

X7 is selected from the group consisting of S, A, C, T, and G or is analiphatic amino acid;

X8 is selected from the group consisting of V, L, I, and M;

X9 is absent or is any amino acid; and

X10 is absent or is any amino acid;

wherein at least one of the following is true;

(a) X3 is N and X7 is not G;

(b) X7 is G and X3 is not N;

(c) X2 is not L;

(d) X4 is not R;

(e) X5 is not Q;

(f) X6 is not L;

(g) X8 is not V;

(h) X10 is absent; or

(i) X9 and X10 are absent.

In addition to the recited amino acids, X2, X3, X6 and X7 can be anyaliphatic amino acid (whether naturally occurring or not), including butnot limited to beta-alanine and 2-aminocyclohexane-1-carboxylic acid.

In various further embodiments, X4 is R; X5 is Q, and/or X8 is V. Invarious further embodiments, X3 is selected from the group consisting ofV, L, I, A, G, Q, and N. In further embodiments, X6 is selected from thegroup consisting of C, A, G, L, V, I, M, Y, W, and F. In various furtherembodiments, X7 is selected from the group consisting of S, A, C, T, andG.

In some embodiments, at least one of Z1 and Z2 are a transductiondomain.

The polypeptides of the present invention are useful, for example, asHSP27 kinase inhibitors, which can be used as therapeutic agents for avariety of disorders, as disclosed in more detail below.

The term “polypeptide” is used in its broadest sense to refer to asequence of subunit amino acids, amino acid analogs, or peptidomimetics.The subunits are linked by peptide bonds, except where noted. Thepolypeptides described herein may be chemically synthesized orrecombinantly expressed.

Preferably, the polypeptides of the present invention are chemicallysynthesized. Synthetic polypeptides, prepared using the well knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (N-α-amino protected N-α-t-butyloxycarbonyl) amino acidresin with the standard deprotecting, neutralization, coupling and washprotocols of the original solid phase procedure of Merrifield (1963, J.Am. Chem. Soc. 85:2149-2154), or the base-labile N-α-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc N-α-aminoprotected amino acids can be obtained from Sigma, Cambridge ResearchBiochemical, or other chemical companies familiar to those skilled inthe art. In addition, the polypeptides can be synthesized with otherN-α-protecting groups that are familiar to those skilled in this art.

Solid phase peptide synthesis may be accomplished by techniques familiarto those in the art and provided, for example, in Stewart and Young,1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co.,Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res.35:161-214, or using automated synthesizers. The polypeptides of theinvention may comprise D-amino acids (which are resistant to L-aminoacid-specific proteases in vivo), a combination of D- and L-amino acids,and various “designer” amino acids (e.g., β-methyl amino acids,C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to conveyspecial properties. Synthetic amino acids include ornithine for lysine,and norleucine for leucine or isoleucine.

In addition, the polypeptides can have peptidomimetic bonds, such asester bonds, to prepare peptides with novel properties. For example, apeptide may be generated that incorporates a reduced peptide bond, i.e.,R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues or sequences. Areduced peptide bond may be introduced as a dipeptide subunit. Such apolypeptide would be resistant to protease activity, and would possessan extended half-live in vivo.

In some embodiments, at least one of Z1 and Z2 is a transduction domain.As used herein, the term “transduction domain” means one or more aminoacid sequence or any other molecule that can carry the active domainacross cell membranes. These domains can be linked to other polypeptidesto direct movement of the linked polypeptide across cell membranes. Insome cases the transducing molecules do not need to be covalently linkedto the active polypeptide. In one embodiment, the transduction domain islinked to the rest of the polypeptide via peptide bonding. (See, forexample, Cell 55: 1179-1188, 1988; Cell 55: 1189-1193, 1988; Proc NatlAcad Sci USA 91: 664-668, 1994; Science 285: 1569-1572, 1999; J BiolChem 276: 3254-3261, 2001; and Cancer Res 61: 474-477, 2001) In afurther embodiment, both Z1 and Z2 are transduction domains. In anotherembodiment, the transduction domain(s) is/are selected from the groupconsisting of (R)₄₋₉ (SEQ ID NO: 1); GRKKRRQRRRPPQ (SEQ ID NO: 2);RQRRKKRG (SEQ ID NO: 3); GRKKRRQR (SEQ ID NO: 4); AYARAAARQARA (SEQ IDNO: 5); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 6);GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 7); PLSSIFSRIGDP (SEQ ID NO: 8);AAVALLPAVLLALLAP (SEQ ID NO: 9); AAVLLPVLLAAP (SEQ ID NO: 10);VTVLALGALAGVGVG (SEQ ID NO: 11); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO: 12);GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 13); KLALKLALKALKAALKLA (SEQ IDNO: 14); KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 15); KAFAKLAARLYRKA (SEQ IDNO: 16); KAFAKLAARLYRAA (SEQ ID NO: 17); AAFAKLAARLYRKA (SEQ ID NO: 18);KAFAALAARLYRKA (SEQ ID NO: 19); KAFAKLAARLYRKAGC (SEQ ID NO: 20);KAFAKLAARLYRAAGC (SEQ ID NO: 21); AAFAKLAARLYRKAGC (SEQ ID NO: 22);KAFAALAARLYRKAGC (SEQ ID NO: 23); KAFAKLAAQLYRKAGC (SEQ ID NO: 24),AGGGGYGRKKRRQRRR (SEQ ID NO: 25); YARAAARQARA (SEQ ID NO: 26);YGRKKRRQRRR (SEQ ID NO: 27); WLRRIKAWLRRIKA (SEQ ID NO: 28);WLRRIKAWLRRIKAWLRRIKA (SEQ ID NO: 29); FAKLAARLYRKA (SEQ ID NO: 30);KAFAALAARLYRKA (SEQ ID NO: 31); KAFAKLAARLYRAA (SEQ ID NO: 32);KAFAKLAARLYRA (SEQ ID NO: 33); FAKLAARLYRAA (SEQ ID NO: 34); andFAKLAARLYRA (SEQ ID NO: 35).

Further exemplary polypeptides according to the invention include, butare not limited to any of those listed above, wherein one or both of Z1and Z2 are selected from the group consisting of WLRRIKAWLRRIKA (SEQ IDNO: 28); WLRRIKAWLRRIKAWLRRIKA (SEQ ID NO: 29); YGRKKRRQRRR (SEQ ID NO:27); YARAAARQARA (SEQ ID NO: 26); RQRRKKRG (SEQ ID NO: 3); GRKKRRQR (SEQID NO: 4); KAFAKLAARLYRKA (SEQ ID NO: 16); FAKLAARLYRKA (SEQ ID NO: 30);KAFAALAARLYRKA (SEQ ID NO: 31); KAFAKLAARLYRAA (SEQ ID NO: 32);KAFAKLAARLYRA (SEQ ID NO: 33); FAKLAARLYRAA (SEQ ID NO: 34); andFAKLAARLYRA (SEQ ID NO: 35).

In various further embodiments, exemplary polypeptides according to theinvention include, but are not limited to those comprising or consistingof:

YARAAARQARAKALARQLGVAA; (SEQ ID NO: 38) YGRKKRRQRRRKALARQLGVAA; (SEQ IDNO: 39) RQRRKKRGKALARQLGVAA; (SEQ ID NO: 40) GRKKRRQRKALARQLGVAA; (SEQID NO: 41) WLRRIKAWLRRIKAKALARQLGVAA; (SEQ ID NO: 42)WLRRIKAWLRRIKAWLRRIKAKALARQLGVAA; (SEQ ID NO: 43)YARAAARQARAKKKALARQLGVAA; (SEQ ID NO: 44) YGRKKRRQRRRKKKALARQLGVAA; (SEQID NO: 45) RQRRKKRGKKKALARQLGVAA; (SEQ ID NO: 46) GRKKRRQRKKKALARQLGVAA;(SEQ ID NO: 47) WLRRIKAWLRRIKAKKKALARQLGVAA; (SEQ ID NO: 48)WLRRIKAWLRRIKAWLRRIKAKKKALARQLG (SEQ ID NO: 49) VAA;KAFAKLAARLYRKALARQLGVAA; (SEQ ID NO: 50) FAKLAARLYRKALARQLGVAA; (SEQ IDNO: 51) KAFAKLAARLYRAALARQLGVAA; (SEQ ID NO: 52) KAFAKLAARLYRALARQLGVAA;(SEQ ID NO: 53) KAFAALAARLYRAALARQLGVAA; (SEQ ID NO: 54)FAKLAARLYRAALARQLGVAA; (SEQ ID NO: 55) WLRRIKAWLRRIKA-LNRQLGVAA; (SEQ IDNO: 56) YARAAARQARAKALNRQLGVA; (SEQ ID NO: 57) KAFAKLAARLYRKALNRQLAVAA;(SEQ ID NO: 58) FAKLAARLYRKALNRQLAVAA; (SEQ ID NO: 59)KAFAKLAARLYRKA-LNRQLGVAA; (SEQ ID NO: 60) FAKLAARLYRKA-LNRQLGVAA; (SEQID NO: 61)

In another aspect, the present invention provides compositions,comprising one or more of the polypeptides disclosed herein, and apharmaceutically acceptable carrier. Such pharmaceutical compositionsare especially useful for carrying out the methods of the inventiondescribed below.

The term “active” refers to the ingredient, component or constituent ofthe compositions of the present invention responsible for the intendedtherapeutic effect.

A “pharmaceutical composition” is one that is employed to prevent,reduce in intensity, cure, or otherwise treat a target condition,syndrome, disorder or disease that has undergone federal regulatoryreview.

As used herein the term “pharmaceutically acceptable carrier” refers toany substantially non-toxic carrier conventionally useable foradministration of pharmaceuticals in which the isolated polypeptide ofthe present invention will remain stable and bioavailable.

For administration, the polypeptides are ordinarily combined with one ormore adjuvants appropriate for the indicated route of administration.The compounds may be admixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,dextran sulfate, glycosaminoglycan-containing gel or non-gelcompositions or coatings, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Examples ofglycosaminoglycans include but are limited to heparin, heparan sulfate,dermatan sulfate, chondroitin sulfate, hyaluronic acid, and keratansulfate. Alternatively, the polypeptides of this invention may bedissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art. The polypeptidesmay be linked to other compounds to promote an increased half-life invivo, such as polyethylene glycol. Such linkage can be covalent ornon-covalent as is understood by those of skill in the art.

The polypeptides may be made up in a solid form (including granules,powders or suppositories) or in a liquid form (e.g., solutions,suspensions, or emulsions). The polypeptides of the invention may beapplied in a variety of solutions. Suitable solutions for use inaccordance with the invention are sterile, dissolve sufficient amountsof the polypeptides, and are not harmful for the proposed application.

In another aspect, the present invention provides an isolated nucleicacid encoding a polypeptide of the present invention. Appropriatenucleic acids according to this aspect of the invention will be apparentto one of skill in the art based on the disclosure provided herein andthe general level of skill in the art.

In another aspect, the present invention provides an expression vectorcomprising DNA control sequences operably linked to the isolated nucleicacids of the present invention, as disclosed above. “Control sequences”operably linked to the nucleic acids of the invention are nucleic acidsequences capable of effecting the expression of the nucleic acids ofthe invention. The control sequences need not be contiguous with thenucleic acids, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the nucleicacid and the promoter sequence can still be considered “operably linked”to the coding sequence. Other such control sequences include, but arenot limited to, polyadenylation signals, termination signals, andribosome binding sites. Such expression vectors can be of any type knownin the art, including but not limited to plasmid and viral-basedexpression vectors.

In a further aspect, the present invention provides geneticallyengineered host cells comprising the expression vectors of theinvention. Such host cells can be prokaryotic cells or eukaryotic cells,and can be either transiently or stably transfected, or can betransduced with viral vectors.

In another aspect, the invention provides biomedical devices comprisingone or more of the polypeptides of the present invention disposed on orin the biomedical device. As used herein, a “biomedical device” refersto a device to be implanted into a subject, for example, a human being,in order to bring about a desired result. Particularly preferredbiomedical devices according to this aspect of the invention include,but are not limited to, stents (including but not limited to coronarystents), grafts (including but not limited to vascular grafts), shunts,stent grafts, fistulas, angioplasty devices, balloon catheters, venouscatheters, implantable drug delivery devices, adhesion barriers(including but not limited to carboxymethylcellulose, hyaluronic acid,and PTFE sheets) to separate tissue, wound dressings such as films(e.g., polyurethane films), hydrocolloids (hydrophilic colloidalparticles bound to polyurethane foam), hydrogels (cross-linked polymerscontaining about at least 60% water), other viscous liquids andhydrogel-like species (including but not limited to, those disclosed inUS 20030190364), foams (hydrophilic or hydrophobic), calcium alginates(nonwoven composites of fibers from calcium alginate), cellophane,pluronics (ie: poly(ethylene glycol)-block-poly(propylene glycol), andbiological polymers.

As used herein, the term “grafts” refers to both natural and prostheticgrafts and implants. In a preferred embodiment, the graft is a vasculargraft.

As used herein, the term “stent” includes the stent itself, as well asany sleeve or other component that may be used to facilitate stentplacement.

As used herein, “disposed on or in” means that the one or morepolypeptides can be either directly or indirectly in contact with anouter surface, an inner surface, or embedded within the biomedicaldevice. “Direct” contact refers to disposition of the polypeptidesdirectly on or in the device, including but not limited to soaking abiomedical device in a solution containing the one or more polypeptides,spin coating or spraying a solution containing the one or morepolypeptides onto the device, implanting any device that would deliverthe polypeptide, and administering the polypeptide through a catheterdirectly on to the surface or into any organ.

“Indirect” contact means that the one or more polypeptides do notdirectly contact the biomedical device. For example, the one or morepolypeptides may be disposed in a matrix, such as a gel matrix (such asa heparin coating) or a viscous fluid, which is disposed on thebiomedical device. Such matrices can be prepared to, for example, modifythe binding and release properties of the one or more polypeptides asrequired. In one non-limiting example, a heparin coating is disposed onthe biomedical device (such as a poly(tetrafluoroethylene) (PTFE)vascular device or sheet) and the one or more polypeptides are disposedon or in a heparin coating; in this example, the one or morepolypeptides can be delivered to a subject in need thereof in acontrolled manner. In one non-limiting example, the release of the oneor more polypeptides from interstitial surfaces ofpoly(tetrafluoroethylene) (PTFE) vascular devices or sheets can becontrolled by first adsorbing or bonding heparin to the surface and/orinterstices of the PTFE device followed by adsorption of polypeptide.Alternating layers of heparin and the polypeptide can also be used toincrease the polypeptide dose and/or time of release. Underphysiological conditions within the body, the kinetics of theassociation and dissociation of polypeptides disclosed herein to andfrom heparin will lead to a delayed release profile as compared torelease of the polypeptide from a bare PTFE device. In addition, therelease profile can be further altered through changes in localtemperature, pH or ionic strength. Such controlled release is of greatvalue for use in the various therapeutic treatments for which thebiomedical devices can be used, as discussed below.

Heparin coatings on various medical devices are known in the art.Applications in humans include central venous catheters, coronarystents, ventricular assist devices, extracorporeal blood circuits, bloodsampling devices, and vascular grafts. Such coatings can be in a gel ornon-gel form. As used herein “heparin coating” includes heparin adsorbedto the surface, heparin bonded to the surface, and heparin imbedded inthe PTFE polymer surface. An example of a method for bonding the heparinwould be to use ammonia plasma to treat, for example, a PTFE surface andreacting the resultant amines with oxidized heparin. Layer-by-layerbuildup of the heparin and one or more polypeptides could then be usedto increase polypeptide on the surface and expand the delivery time. Gelforms of the heparin coating can include, but are not limited to, anyhydrogel containing heparin either covalently or physically bound to thegel. The heparin coating is disposed on the biomedical device, whichincludes direct contact with an outer surface or an inner surface of thebiomedical device, or embedded within the biomedical device. “Direct”contact refers to disposition directly on or in the device, includingbut not limited to soaking a biomedical device in a heparin coatingsolution (wherein the polypeptides may be added as part of the heparincoating solution, or may be subsequently disposed on or in the heparincoating after it is contacted with the device), spin coating or sprayinga heparin coating solution onto the device (wherein the polypeptides maybe added as part of the heparin coating solution, or may be subsequentlydisposed on or in the heparin coating after it is contacted with thedevice), and administering the heparin coating solution containing thepolypeptides through a catheter directly on to the surface or into anyorgan. The physical characteristics and specific composition of theheparin layer can be any that provides the desired release profile ofthe one or more polypeptides. See, for example, Seal and Panitch,Biomacromolecules 2003(4):1572-1582 (2003); US20030190364, incorporatedby reference herein in its entirety; and Carmeda BioActive Surface(CBAS™) the product of Carmeda AB in Stockholm, Sweden. “Indirect”contact means that the heparin coating is not directly in contact withthe device such as, for example, when an intervening coating is placedbetween the device surface and the heparin coating. In one non-limitingexample, the one or more polypeptides could be initially adsorbed(directly or indirectly), and then adsorbing a heparin coating; this canoptionally be followed by subsequent polypeptide layers, heparin layers,or combinations thereof, as desired. As will be understood by those ofskill in the art, any sulfated polysaccharide or negatively chargedpolymer can be used in like manner to heparin as described above, toprovide desired release characteristics.

In a further aspect, the present invention provides methods for one ormore of the following therapeutic uses: (a) reducing smooth muscle cellproliferation and/or migration; (b) promoting smooth muscle relaxation;(c) increasing the contractile rate in heart muscle; (d) increasing therate of heart muscle relaxation; (e) promoting wound healing; (f)treating and/or reducing fibrotic disorders and/or keloids; (g) reducingscar formation; (h) disrupting focal adhesions; (i) regulating actinpolymerization; and (j) treating or reducing incidence of one or more ofintimal hyperplasia, stenosis, restenosis, atherosclerosis, smoothmuscle cell tumors and metastasis, smooth muscle spasm, angina,Prinzmetal's angina, ischemia, stroke, bradycardia, hypertension,cardiac hypertrophy, renal failure, stroke, pulmonary hypertension,asthma, toxemia of pregnancy, pre-term labor, pre-eclampsia/eclampsia,Raynaud's disease or phenomenon, hemolytic-uremia, non-occlusivemesenteric ischemia, anal fissure, achalasia, impotence, migraine,ischemic muscle injury associated with smooth muscle spasm,vasculopathy, bradyarrythmia, bradycardia, congestive heart failure,stunned myocardium, pulmonary hypertension, diastolic dysfunction,gliosis (proliferation of astrocytes, and may include deposition ofextracellular matrix, including but not limited to such proliferationand extracellular matrix (“ECM”) deposition in damaged areas of thecentral nervous system); chronic obstructive pulmonary disease (i.e.,respiratory tract diseases characterized by airflow obstruction orlimitation; includes but is not limited to chronic bronchitis andemphysema), osteopenia, endothelial dysfunction, inflammation,rheumatoid arthritis, degenerative arthritis, ankylosing spondylitis,Sjogren's disease, Guilliame-Barre disease, infectious disease, sepsis,endotoxemic shock, psoriasis, radiation enteritis, scleroderma,cirrhosis, interstitial fibrosis, Crohn's disease, colitis, inflammatorybowel disease, appendicitis, gastritis, laryngitis, meningitis,pancreatitis, otitis, reperfusion injury, traumatic brain injury, spinalcord injury, peripheral neuropathy, multiple sclerosis, Lupus, allergy,cardiometabolic diseases, cardiovascular diseases, obesity, type IIdiabetes mellitus, type I diabetes mellitis, NASH/cirrhosis, andAlzheimer's disease; wherein the method comprises administering to asubject in need thereof an effective amount to carry out the one or moretherapeutic uses of one or more polypeptides or compositions accordingto the present invention, or functional equivalents thereof.

Without being bound by theory, it is believed that the polypeptides ofthe present invention provide their therapeutic effect as a result ofinhibiting HSP27 phosphorylation by HSP27 kinase (MAPKAP kinase 2),although alternative mechanisms, including but not limited to inhibitionof HSP27 phosphorylation by MAPKAP kinase 3, and MAPKAP kinase 5 arealso encompassed by the present invention.

Since MAPKAP2 is downstream of p38 MAP kinase, any therapeutic uses forwhich p38 MAPK inhibitors are useful are within the scope of the presentinvention as well.

As used herein, “treat” or “treating” means accomplishing one or more ofthe following: (a) reducing the severity of the disorder; (b) limitingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) limiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting recurrence ofsymptoms in patients that were previously symptomatic for thedisorder(s).

As used herein, the term “reduce” or “reducing” means to limitoccurrence of the disorder in individuals at risk of developing thedisorder.

As used herein, “administering” includes in vivo administration, as wellas administration directly to tissue ex vivo, such as vein grafts. Thecompositions of the present invention may be administered systemicallyeither orally, buccally, parenterally, topically, by inhalation orinsufflation (i.e., through the mouth or through the nose), or rectallyin dosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants, and vehicles asdesired.

For buccal administration, the compositions of the present invention maytake the form of tablets or lozenges formulated in a conventionalmanner.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle); intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord), intrasternal injection, or infusiontechniques. A parenterally administered composition of the presentinvention is delivered using a needle, e.g., a surgical needle. The term“surgical needle” as used herein, refers to any needle adapted fordelivery of fluid (i.e., capable of flow) compositions of the presentinvention into a selected anatomical structure. Injectable preparations,such as sterile injectable aqueous or oleaginous suspensions, may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents.

The sterile injectable preparation also may be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. A solutiongenerally is considered as a homogeneous mixture of two or moresubstances; it is frequently, though not necessarily, a liquid. In asolution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. A suspension is adispersion (mixture) in which a finely-divided species is combined withanother species, with the former being so finely divided and mixed thatit doesn't rapidly settle out. In everyday life, the most commonsuspensions are those of solids in liquid water. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. Forparenteral application, particularly suitable vehicles consist ofsolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants. Aqueous suspensions may contain substances whichincrease the viscosity of the suspension and include, for example,sodium carboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers.

The term “topical” refers to administration of an inventive compositionat, or immediately beneath, the point of application. The phrase“topically applying” describes application onto one or more surfaces(s)including epithelial surfaces. Although topical administration, incontrast to transdermal administration, generally provides a localrather than a systemic effect, as used herein, unless otherwise statedor implied, the terms topical administration and transdermaladministration are used interchangeably.

Topical administration also may involve the use of transdermaladministration such as transdermal patches or iontophoresis deviceswhich are prepared according to techniques and procedures well known inthe art. The terms “transdermal delivery system”, transdermal patch” or“patch” refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for post-menopausalindications, and nicotine for smoking cessation. Patches suitable foruse in the present invention include, but are not limited to, (1) thematrix patch; (2) the reservoir patch; (3) the multi-laminatedrug-in-adhesive patch; and (4) the monolithic drug-in-adhesive patch;TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS, pp. 249-297 (Tapash K.Ghosh et al. eds., 1997), hereby incorporated herein by reference. Thesepatches are well known in the art and generally available commercially.

The compositions of the present invention may be in the form of adispersible dry powder for delivery by inhalation or insufflation(either through the mouth or through the nose). Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures ofwhich are incorporated by reference. The composition of the presentinvention is placed within a suitable dosage receptacle in an amountsufficient to provide a subject with a unit dosage treatment. The dosagereceptacle is one that fits within a suitable inhalation device to allowfor the aerosolization of the dry powder composition by dispersion intoa gas stream to form an aerosol and then capturing the aerosol soproduced in a chamber having a mouthpiece attached for subsequentinhalation by a subject in need of treatment. Such a dosage receptacleincludes any container enclosing the composition known in the art suchas gelatin or plastic capsules with a removable portion that allows astream of gas (e.g., air) to be directed into the container to dispersethe dry powder composition. Such containers are exemplified by thoseshown in U.S. Pat. Nos. 4,227,522; 4,192,309; and 4,105,027. Suitablecontainers also include those used in conjunction with Glaxo's Ventolin®Rotohaler brand powder inhaler or Fison's Spinhaler® brand powderinhaler. Another suitable unit-dose container which provides a superiormoisture barrier is formed from an aluminum foil plastic laminate. Thepharmaceutical-based powder is filled by weight or by volume into thedepression in the formable foil and hermetically sealed with a coveringfoil-plastic laminate. Such a container for use with a powder inhalationdevice is described in U.S. Pat. No. 4,778,054 and is used with Glaxo'sDiskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237). All ofthese references are incorporated herein by reference.

The compositions of the present invention may be in the form ofsuppositories for rectal administration of the composition. “Rectal” or“rectally” as used herein refers to introduction into the body throughthe rectum where absorption occurs through the walls of the rectum.These compositions can be prepared by mixing the drug with a suitablenonirritating excipient such as cocoa butter and polyethylene glycolswhich are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.When formulated as a suppository the compositions of the invention maybe formulated with traditional binders and carriers, such astriglycerides.

The following are nonlimiting examples of routes of delivery for variousindications of the different embodiments of the methods of theinvention: topical administration may be used for methods involvingtreatment or reducing the incidence of vein graft spasm, intimalhyperplasia, restenosis, prosthetic graft failure due to intimalhyperplasia, stent, stent graft failure due to intimalhyperplasia/constrictive remodeling, microvascular graft failure due tovasospasm, transplant vasculopathy, scarring, fibrosis, keloidformation, male and female sexual dysfunction, prevention ofhydrocephalus caused by subarachnoid hemorrhage, and for promoting woundhealing; intrathecal administration may be used for treating or reducingincidence of stroke and subarachnoid hemorrhage induced vasospasm;intraperitoneal administration may be used for treating or reducingincidence of non-occlusive mesenteric ischemia; oral administration maybe used for treating or reducing incidence of achalasia; intravenousadministration may be used for treating or reducing incidence ofhypertension and bradycardia; rectal administration may be used fortreating or reducing incidence of anal fissure; aerosol delivery may beused for treating or reducing incidence of asthma (ie: bronchospasm);and intrauterine administration may be used for treating or reducingincidence of pre-term labor and pre-eclampsia/eclampsia.

The term “disease” or “disorder”, as used herein, refers to animpairment of health or a condition of abnormal functioning. The term“syndrome,” as used herein, refers to a pattern of symptoms indicativeof some disease or condition. The term “injury,” as used herein, refersto damage or harm to a structure or function of the body caused by anoutside agent or force, which may be physical or chemical. The term“condition”, as used herein, refers to a variety of health states and ismeant to include disorders or diseases caused by any underlyingmechanism or disorder, injury, and the promotion of healthy tissues andorgans.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “subject” or “individual” are used interchangeably to refer toa member of an animal species of mammalian origin, including humans.

Intimal hyperplasia is a complex process that leads to graft failure,and is the most common cause of failure of arterial bypass grafts. Whileincompletely understood, intimal hyperplasia is mediated by a sequenceof events that include endothelial cell injury and subsequent vascularsmooth muscle proliferation and migration from the media to the intima.This process is associated with a phenotypic modulation of the smoothmuscle cells from a contractile to a synthetic phenotype. The“synthetic” smooth muscle cells secrete extracellular matrix proteins,which leads to pathologic narrowing of the vessel lumen leading to graftstenoses and ultimately graft failure. Such endothelial cell injury andsubsequent smooth muscle cell proliferation and migration into theintima also characterize restenosis, most commonly after angioplasty toclear an obstructed blood vessel.

In some embodiments of the methods of the invention, such as thoserelating to reducing occurrence of smooth muscle cell proliferationand/or migration, or promoting smooth muscle relaxation, theadministering may be direct, by contacting a blood vessel in a subjectbeing treated with one or more polypeptides of the invention. Forexample, a liquid preparation of one or more polypeptides according tothe invention can be forced through a porous catheter, or otherwiseinjected through a catheter to the injured site, or a gel or viscousliquid containing the one or more polypeptides according to theinvention can be spread on the injured site. In these embodiments ofdirect delivery, one or more polypeptides according to the invention isdelivered into smooth muscle cells at the site of injury orintervention. This can be accomplished, for example, by delivering therecombinant expression vectors (most preferably a viral vector, such asan adenoviral vector) of the invention to the site. Alternatively,delivery into smooth muscle cells is accomplished by using the one ormore polypeptides according to the invention that include at least onetransduction domain to facilitate entry into the smooth muscle cells.

In various other embodiments of the methods of the invention,particularly those that involve reducing occurrence of smooth musclecell proliferation and/or migration, the method is performed on asubject who has undergone, is undergoing, or will undergo a procedureselected from the group consisting of angioplasty, vascular stentplacement, endarterectomy, atherectomy, bypass surgery (such as coronaryartery bypass surgery; peripheral vascular bypass surgeries), vasculargrafting, organ transplant, prosthetic device implanting, microvascularreconstructions, plastic surgical flap construction, and catheteremplacement.

In another embodiment, the methods comprise treating or reducingoccurrence of one or more disorders selected from the group consistingof intimal or neointimal hyperplasia, stenosis, restenosis, andatherosclerosis, comprising contacting a subject in need thereof with anamount effective to treat or reduce intimal or neointimal hyperplasia,stenosis, restenosis, and/or atherosclerosis of one or more polypeptidesaccording to the invention.

In a further embodiment of this aspect of the invention, the method isused to treat tumors and/or metastasis, including but not limited tosmooth muscle tumors. In one embodiment, the tumor is a leiomyosarcoma,which is defined as a malignant neoplasm that arises from muscle. Sinceleiomyosarcomas can arise from the walls of both small and large bloodvessels, they can occur anywhere in the body, but peritoneal, uterine,and gastro-intestinal (particularly esophageal) leiomyosarcomas are morecommon. Alternatively, the smooth muscle tumor can be a leiomyoma, anon-malignant smooth muscle neoplasm. In a further embodiment, themethod can be combined with other treatments for smooth muscle celltumors and/or metastasis, such as chemotherapy, radiation therapy, andsurgery to remove the tumor. Without being limited by theory,administration of the polypeptides of the invention can be used to treattumors and/or metastasis by any or all of the following mechanisms:preventing drug resistance to anticancer drugs or promotingsusceptibility to anti cancer drugs as a kinase inhibitor, promotingapoptosis of cancer cells, decreasing cell invasion through decreasedmatrix metalloproteinase expression and decreased migration of cancercells, and through suppressing viral oncogenesis.

In a further embodiment, the methods of the invention are used fortreating or reducing occurrence of smooth muscle spasm, comprisingcontacting a subject or graft in need thereof with an amount effectiveto reduce smooth muscle spasm of one or more polypeptides according tothe invention.

Smooth muscles are found in the walls of blood vessels, airways, thegastrointestinal tract, and the genitourinary tract. Pathologic toniccontraction of smooth muscle constitutes spasm. Many pathologicalconditions are associated with spasm of vascular smooth muscle(“vasospasm”), the smooth muscle that lines blood vessels. This cancause symptoms such as angina and ischemia (if a heart artery isinvolved), or stroke as in the case of subarachnoid hemorrhage inducedvasospasm if a brain vessel is involved. Hypertension (high bloodpressure) is caused by excessive vasoconstriction, as well asthickening, of the vessel wall, particularly in the smaller vessels ofthe circulation.

Thus, in a further embodiment of the methods of the invention, themuscle cell spasm comprises a vasospasm, and the methods of theinvention are used to treat or reduce occurrence of vasospasm.Embodiments of the method include, but are not limited to, methods totreat or inhibit angina, coronary vasospasm, Prinzmetal's angina(episodic focal spasm of an epicardial coronary artery), ischemia,stroke, bradycardia, and hypertension.

In another embodiment of the methods of the invention, occurrence ofsmooth muscle spasm is reduce by treatment of a graft, such as a vein orarterial graft, with the one or more polypeptides according to theinvention. One of the ideal conduits for peripheral vascular andcoronary reconstruction is the greater saphenous vein. However, thesurgical manipulation during harvest of the conduit often leads tovasospasm. The exact etiology of vasospasm is complex and most likelymultifactorial. Most investigations have suggested that vasospasm iseither due to enhanced constriction or impaired relaxation of thevascular smooth muscle in the media of the vein. Numerousvasoconstricting agents such as endothelin-1 and thromboxane areincreased during surgery and result in vascular smooth musclecontraction. Other vasoconstrictors such as norepinephrine,5-hydroxytryptamine, acetylcholine, histamine, angiotensin II, andphenylephrine have been implicated in vein graft spasm. Papaverine is asmooth muscle vasodilator that has been used. In circumstances wherespasm occurs even in the presence of papaverine, surgeons useintraluminal mechanical distension to break the spasm. This leads toinjury to the vein graft wall and subsequent intimal hyperplasia.Intimal hyperplasia is the leading cause of graft failure.

Thus, in this embodiment, the graft can be contacted with the one ormore polypeptides according to the invention, during harvest from thegraft donor, subsequent to harvest (before implantation), and/or duringimplantation into the graft recipient (ie: ex vitro or in vivo). Thiscan be accomplished, for example, by delivering the recombinantexpression vectors (most preferably a viral vector, such as anadenoviral vector) of the invention to the site, and transfecting thesmooth muscle cells. Alternatively, delivery into smooth muscle isaccomplished by using the one or more polypeptides according to theinvention that include at least one transduction domain to facilitateentry into the smooth muscle cells. In some embodiments, during graftimplantation, the subject receiving the graft is treated systemicallywith heparin, as heparin has been shown to bind to protein transductiondomains and prevent them from transducing into cells. This approach willlead to localized protein transduction of the graft alone, and not intoperipheral tissues. The methods of this embodiment of the inventionreduce occurrence of vein graft spasm during harvest and/or implantationof the graft, and thus improve both short and long term graft success.

In various other embodiments of the methods of the invention, the musclecell spasm is associated with a disorder including, but not limited topulmonary (lung) hypertension, asthma (bronchospasm), toxemia ofpregnancy, pre-term labor, pre-eclampsia/eclampsia, Raynaud's disease orphenomenon, hemolytic-uremia, non-occlusive mesenteric ischemia(ischemia of the intestines that is caused by inadequate blood flow tothe intestines), anal fissure (which is caused by persistent spasm ofthe internal anal sphincter), achalasia (which is caused by persistentspasm of the lower esophageal sphincter), impotence (which is caused bya lack of relaxation of the vessels in the penis, erection requiresvasodilation of the corpra cavernosal (penile) blood vessels), migraine(which is caused by spasm of the intracranial blood vessels), ischemicmuscle injury associated with smooth muscle spasm, and vasculopathy,such as transplant vasculopathy (a reaction in the transplanted vesselswhich is similar to atherosclerosis, it involves constrictive remodelingand ultimately obliteration of the transplanted blood vessels, this isthe leading cause of heart transplant failure).

In other embodiments, the methods of the invention are used for one ormore of promoting wound healing, reducing scar formation, treatingand/or reducing fibrotic disorders and treating and/or reducing keloids.In these embodiments, an “individual in need thereof” is an individualthat has suffered or will suffer (for example, via a surgical procedure)a wound that may result in scar formation, or has resulted in scarformation. As used herein, the term “wound” refers broadly to injuriesto the skin and subcutaneous tissue. Such wounds include, but are notlimited to lacerations; burns; punctures; pressure sores; bed sores;canker sores; trauma, bites; fistulas; ulcers; lesions caused byinfections; periodontal wounds; endodontic wounds; burning mouthsyndrome; laparotomy wounds; surgical wounds; incisional wounds;contractures after burns; tissue fibrosis, including but not limited toidiopathic pulmonary fibrosis, hepatic fibrosis, renal fibrosis,retroperitoaneal fibrosis, and cystic fibrosis, but excluding bloodvessel fibrosis or heart tissue fibrosis; and wounds resulting fromcosmetic surgical procedures. In these embodiments, the one or morepolypeptides or compositions are disposed on or in a wound dressing orother topical administration. Such wound dressings can be any used inthe art, including but not limited to films (e.g., polyurethane films),hydrocolloids (hydrophilic colloidal particles bound to polyurethanefoam), hydrogels (cross-linked polymers containing about at least 60%water), foams (hydrophilic or hydrophobic), calcium alginates (nonwovencomposites of fibers from calcium alginate), cellophane, and biologicalpolymers such as those described in US patent application publicationnumber 20030190364, published Oct. 9, 2003, which is incorporated hereinby reference.

As used herein, the phrase “reducing scar formation” means any decreasein scar formation that provides a therapeutic or cosmetic benefit to thepatient. Such a therapeutic or cosmetic benefit can be achieved, forexample, by decreasing the size and/or depth of a scar relative to scarformation in the absence of treatment with the methods of the invention,or by reducing the size of an existing scar. As used herein, such scarsinclude scars of all types, including but not limited to keloids;hypertrophic scars; and adhesion formation between organ surfaces,including but not limited to those occurring as a result of surgery.

The methods of these embodiments are clinically useful for treating alltypes of wounds to reduce scar formation, both for reducing initial scarformation, and for therapeutic treatment of existing scars (i.e.:cutting out the scar after its formation, treating it with the compoundsof the invention, and letting the scar heal more slowly). In someembodiments, individuals in need of treatment or limiting of scarring(such as keloids or hypertrophic scarring) are highly pigmentedindividuals, including but not limited to individuals of Asian orAfrican descent, that are susceptible to keloids, and thus can benefitfrom the methods of the invention for prophylactic therapy to limitdevelopment of keloids, as well as for treating keloids. In variousother embodiments, individuals in need of therapy for treating orlimiting fibrotic disorders are those suffering from or at risk of oneor more fibrotic disorders associated with TGFβ-induced CTGF expression,including but not limited to tissue fibrosis (including but not limitedto idiopathic pulmonary fibrosis, hepatic fibrosis, renal fibrosis,retroperitoneal fibrosis, cystic fibrosis, blood vessel fibrosis, CNSfibrosis, and heart tissue fibrosis); diabetic nephropathy,glomerulosclerosis, and IgA nephropathy (causes of kidney failure andthe need for dialysis and retransplant); diabetic retinopathy andmacular degeneration (fibrotic diseases of the eye and leading causes ofblindness); cirrhosis and biliary atresia (leading causes of liverfibrosis and failure); congestive heart failure; lung fibrosis;scleroderma; abdominal adhesions; and interstitial fibrosis.

In various other embodiments, individuals in need of therapy fortreating and/or limiting fibrotic disorders and/or keloids are thosewith elevated levels of one or more of the following biomarkers:

-   -   TGFβ1 expression;    -   Collagen I;    -   CTGF expression; and    -   alpha smooth muscle actin.

Elevated levels of such biomarkers can be detected using standardtechniques, including but not limited to immunological techniques(ELISA, immunocytochemistry, etc.) using commercially availableantibodies against the one or more biomarkers

As disclosed below, the polypeptides of the invention inhibitTGFβ1-induced CTGF and collagen expression in human keloid fibroblasts,which are elevated in fibrotic conditions, indicating that individualswith elevated levels of one or more of these biomarkers can especiallybenefit from the methods of the present invention. As used herein, an“elevated” level of the one or more biomarkers means any increase abovenormal for that individual or similarly situated individuals in arelevant target tissue. Such target tissues are those affected byfibrotic conditions, including but not limited to blood, wound exudate,and biopsies taken from tissues affected by fibrosis including but notlimited to those disclosed above (skin, kidney, lung, liver, peritoneum,blood vessel, heart, retina, etc.) In various further embodiments, anindividual in need thereof is one that has a level of one or more of therecited biomarkers 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, or more abovenormal levels. Determining the level of the one or more biomarkers canbe done using standard techniques in the art for measuring proteinand/or gene expression, including but not limited to those disclosedbelow.

A “normal” level of these one or more biomarkers may be established byany suitable means, including but not limited to determining a normallevel in that individual or similarly situated individuals in theabsence of fibrotic conditions and/or keloids, or any other suitablemeans to establish a standard for reference. A method to treat adisease, disorder or condition according to the present inventioncomprises the steps of (1) administering to a subject in need thereof atherapeutically effective amount of a composition according to thepresent invention; (2) monitoring a level of at least one biomarker in atarget tissue, wherein the at least one biomarker is selected from thegroup consisting of:

-   -   TGFβ1 expression;    -   collagen I expression;    -   CTGF expression; and    -   α-smooth muscle actin expression;        and (3) maintaining the level of the biomarker in the target        tissue substantially at normal levels during treatment.

In another embodiment of the methods of the invention, the methods areused to increase the contractile rate in heart muscle. Individuals thatcan benefit from such treatment include those who exhibit a reducedheart rate relative to either a normal heart rate for the individual, orrelative to a “normal” heart rate for a similarly situated individual.As used herein, the phrase “increasing the contractile rate in heartmuscle” means any increase in contractile rate that provides atherapeutic benefit to the patient. Such a therapeutic benefit can beachieved, for example, by increasing the contractile rate to make itcloser to a normal contractile rate for the individual, a normalcontractile rate for a similarly situated individual, or some otherdesired target contractile rate. In a one embodiment, the methods resultin an increase of at least 5% in the contractile rate of the patient inneed of such treatment. In further embodiments, the methods of theinvention result in an increase of at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, and/or 50% in the contractile rate of the patient in needof such treatment. In some embodiments, increasing the contractile ratein heart muscle is accomplished by increasing the heart musclerelaxation rate (i.e., if the muscles relax faster, they beat faster).In other embodiments, the methods of the invention result in an increaseof at least 5% in the heart muscle relaxation rate of the patient inneed of such treatment. In further embodiments, the methods of theinvention result in an increase of at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, and/or 50% in the heart muscle relaxation rate of thepatient in need of such treatment.

In a further embodiment of the methods of the invention, the methods areperformed to treat one or more cardiac disorders that can benefit fromincreasing the contractile rate in heart muscle. Such cardiac disordersinclude bradyarrythmias, bradycardias congestive heart failure,pulmonary hypertension, stunned myocardium, and diastolic dysfunction.As used herein, “bradyarrythmia” means an abnormal decrease of the rateof the heartbeat to less than 60 beats per minute, generally cased by adisturbance in the electrical impulses to the heart. A common cause ofbradyarrythmias is coronary heart disease, which leads to the formationof atheromas that limit the flow of blood to the cardiac tissue, andthus the cardiac tissue becomes damaged. Bradyarrythmias due to coronaryartery disease occur more frequently after myocardial infarction.Symptoms include, but are not limited to, loss of energy, weakness,syncope, and hypotension.

As used herein, “Congestive heart failure” means an inability of theheart to pump adequate supplies of blood throughout the body. Such heartfailure can be due to a variety of conditions or disorders, includingbut not limited to hypertension, anemia, hyperthyroidism, heart valvedefects including but not limited to aortic stenosis, aorticinsufficiency, and tricuspid insufficiency; congenital heart defectsincluding but not limited to coarctation of the aorta, septal defects,pulmonary stenosis, and tetralogy of Fallot; arrythmias, myocardialinfarction, cardiomyopathy, pulmonary hypertension, and lung diseaseincluding but not limited to chronic bronchitis and emphysema. Symptomsof congestive heart failure include, but are not limited to, fatigue,breathing difficulty, pulmonary edema, and swelling of the ankles andlegs.

As used herein, “Stunned myocardium” means heart muscle that is notfunctioning (pumping/beating) due to cardiac ischemia (lack of bloodflow/oxygen to the vessels supplying the heat muscle).

As used herein, “Diastolic dysfunction” means an inability of the heartto fill with blood during diastole (the resting phase of heartcontraction). This condition usually occurs in the setting of leftventricular hypertrophy. The heart muscle becomes enlarged and stiffsuch that it cannot fill adequately. Diastolic dysfunction can result inheart failure and inadequate heart function.

As used herein, “Pulmonary hypertension” means a disorder in which theblood pressure in the arteries supplying the lungs is abnormally high.Causes include, but are not limited to, inadequate supply of oxygen tothe lungs, such as in chronic bronchitis and emphysema; pulmonaryembolism, and intestinal pulmonary fibrosis. Symptoms and signs ofpulmonary hypertension are often subtle and nonspecific. In the laterstages, pulmonary hypertension leads to right heart failure that isassociated with liver enlargement, enlargement of veins in the neck andgeneralized edema.

In a further embodiment of the methods of the invention, the methods areused for treating a heart muscle disorder comprising administering to anindividual suffering from one or more of bradyarrythmia, bradycardia,congestive heart failure, stunned myocardium, pulmonary hypertension,and diastolic dysfunction, an amount effective to increase heart musclecontractile rate of one or more polypeptides according to the presentinvention.

Treating bradyarrythmia includes one or more of the following (a)improving the rate of the heartbeat to, closer to normal levels for theindividual, closer to a desired rate, or increasing to at least above 60beats per minute; (b) reducing the occurrence of one or more of loss ofenergy, weakness, syncope, and hypotension in patients suffering frombradyarrythmia; (c) reducing worsening of one or more of loss of energy,weakness, syncope, and hypotension in patients suffering frombradyarrythmia and its symptoms; (d) reducing recurrence ofbradyarrythmia in patients that previously suffered from bradyarrythmia;and (e) reducing recurrence of one or more of loss of energy, weakness,syncope, and hypotension in patients that previously suffered frombradyarrythmia.

Similarly, treating congestive heart failure includes one or more of thefollowing (a) improving the heart's ability to pump adequate supplies ofblood throughout the body to closer to normal levels for the individual,or closer to a desired pumping capacity; (b) reducing development of oneor more of fatigue, breathing difficulty, pulmonary edema, and swellingof the ankles and legs in patients suffering from congestive heartfailure; (c) reducing worsening of one or more of fatigue, breathingdifficulty, pulmonary edema, and swelling of the ankles and legs inpatients suffering from congestive heart failure and its symptoms; (d)reducing recurrence of congestive heart failure in patients thatpreviously suffered from congestive heart failure; and (e) reducingrecurrence of one or more of fatigue, breathing difficulty, pulmonaryedema, and swelling of the ankles and legs in patients that previouslysuffered from congestive heart failure.

Treating stunned myocardium means one or more of (a) improving theability of the heart muscle to pump by improving the oxygenation of theischemic muscle, or by decreasing the need of the myocardial cells foroxygen and (b) reducing recurrence of stunned myocardium in patientsthat previously suffered from stunned myocardium.

Similarly, treating diastolic dysfunction includes one or more of (a)reducing occurrence of heart failure and/or inadequate heart function byallowing the heart to relax and fill more completely; (b) reducingrecurrence of diastolic dysfunction in patients that previously sufferedfrom diastolic dysfunction; and (c) reducing recurrence of heart failureand/or inadequate heart function in patients that previously sufferedfrom diastolic dysfunction.

Treating pulmonary hypertension includes one or more of the following(a) decreasing blood pressure in the arteries supplying the lungs tocloser to normal levels for the individual, or closer to a desiredpressure; (b) reducing the occurrence of one or more of enlargement ofveins in the neck, enlargement of the liver, and generalized edema inpatients suffering from pulmonary hypertension; (c) reducing worseningof one or more of enlargement of veins in the neck, enlargement of theliver, and generalized edema in patients suffering from pulmonaryhypertension and its symptoms; (d) reducing recurrence of pulmonaryhypertension in patients that previously suffered from pulmonaryhypertension; and (e) reducing recurrence of one or more of enlargementof veins in the neck, enlargement of the liver, and generalized edema inpatients that previously suffered from pulmonary hypertension.

In a further aspect, the present invention provides methods for reducingoccurrence of a heart muscle disorder comprising administering to anindividual at risk of developing bradyarrythmia, bradycardia, congestiveheart failure, stunned myocardium, pulmonary hypertension, and diastolicdysfunction an amount effective to increase heart muscle contractilerate of one or more polypeptides or compositions according to thepresent invention.

For example, methods to reduce occurrence of congestive heart failureinvolve administration of one or more polypeptides or compositionsaccording to the present invention to a subject that suffers from one ormore of hypertension, anemia, hyperthyroidism, heart valve defectsincluding but not limited to aortic stenosis, aortic insufficiency, andtricuspid insufficiency; congenital heart defects including but notlimited to coarctation of the aorta, septal defects, pulmonary stenosis,and tetralogy of Fallot; arrythmias, myocardial infarction,cardiomyopathy, pulmonary hypertension, and lung disease including butnot limited to chronic bronchitis and emphysema.

Similarly, methods to reduce occurrence of bradyarrythmia involveadministration of the one or more polypeptides or compositions accordingto the present invention to a subject that suffer from one or more ofcoronary heart disease and atheroma formation, or that previously had amyocardial infarction or conduction disorder.

Similarly, methods to reduce occurrence of pulmonary hypertensioninvolve administration of the one or more polypeptides or compositionsaccording to the present invention to a subject that suffers from one ormore of chronic bronchitis, emphysema, pulmonary embolism, andintestinal pulmonary fibrosis.

Reducing occurrence of stunned myocardium involves administration of theone or more polypeptides or compositions according to the presentinvention to a subject that suffers from cardiac ischemia.

Reducing occurrence of or treating diastolic dysfunction involvesadministration of the one or more polypeptides or compositions accordingto the present invention to a subject that suffers from left ventricularhypertrophy

In other embodiments, the methods of the invention are used to treat orlimit the incidence of inducing neural regeneration for central nervoussystem injuries. As used herein, the term “central nervous system” (CNS)refers to the brain and spinal cord. As used herein, the term “neuralregeneration” includes both regenerating a damaged neural connection, aswell as promoting an increase in neural function (including but notlimited to treatment of Alzheimer's and peripheral neuropathy); suchneural regeneration can be in peripheral nervous system or the centralnervous system. Without being limited by theory, it is believed thatadministration of the peptides to a patient in need thereof prevents orlimits activity of the protein rho, which is known to cause growth conecollapse; thus, minimizing rho activity enhances neurite outgrowth.

In other embodiments, the methods of the invention are used to treat orlimit the incidence of gliosis (proliferation of astrocytes in damagedareas of the central nervous system). Astrocytes are the connectivetissue cells of the CNS, and have functions including accumulating inareas with damaged neurons. Gliosis occurs during any traumatic braininjury, insertion of neural electrodes and during spinal cord injury, aswell as in various neurodegenerative disorders including but not limitedto Korsakoff's syndrome and AIDS dementia complex. Without being limitedby theory, it is believed that administration of the peptides to apatient in need thereof prevents or limits the fibrotic response ofastrocytes and possibly microglia to inhibit fibrosis.

In other embodiments, the methods of the invention are used to treat orlimit the incidence of chronic obstructive pulmonary disease (COPD),which is a group of respiratory tract diseases characterized by airflowobstruction or limitation. COPD can be caused by a variety of factors,including but not limited to tobacco smoking (chronic smokers at risk),exposure to coal dust (coal mining industry workers particularly atrisk), congenital defects (including but not limited to alpha1-antitrypsin deficiency), or it may be idiopathic (no known cause).COPD includes, but is not limited to chronic bronchitis and emphysema.Symptoms characteristic of COPD (for which the methods of the inventioncan be used to treat or reduce incidence of) include, but are notlimited to recurrent respiratory infections, severe cough, constantwheezing, shortness of breath with minimal exertion or rest, hypoxia,and excessive sputum production.

The polypeptides of the invention can be used alone or together withother treatments for COPD, including, bronchodilators, antibiotics, andoral or intravenous steroids.

In other embodiments, the methods of the invention are used to treat orlimit the incidence of inflammation. As used herein, “inflammation”means the response by the immune system to infection, irritation, orassociated with foreign bodies (introduction of biomaterials) in thebody.

In various other embodiments, individuals in need of therapy fortreating and/or limiting inflammatory disorders and/or autoimmunediseases oftentimes are those with elevated levels of one or more of thefollowing biomarkers:

-   -   TGFβ1 expression;    -   TNF-α;    -   IL-1;    -   IL-6;    -   IL-8;    -   COX-2;    -   MIP-1α; and    -   MIP-2.

Elevated levels of such biomarkers can be detected using standardtechniques, including but not limited to immunological techniques(ELISA, immunocytochemistry, etc.) using commercially availableantibodies against the one or more biomarkers.

Symptoms characteristic of inflammation (for which the methods of theinvention can be used to treat or reduce incidence of) include, but arenot limited to redness, heat, swelling, pain, and dysfunction of theorgans involved. Specific inflammatory disorders that can be treated, orwhose incidence can be reduced, by the methods of the invention include,but are not limited to, asthma, arthritis (rheumatoid or degenerative),sepsis, endotoxemic shock, psoriasis, radiation enteritis, scleroderma,cirrhosis, interstitial fibrosis, Crohn's disease, inflammatory boweldisease, appendicitis, gastritis, laryngitis, meningitis, pancreatitis,and otitsis.

Without being limited by theory, it is believed that administration ofthe polypeptides of the invention to a patient in need ofanti-inflammatory treatment suppresses the response to and/or expressionof inflammatory cytokines including but not limited to TGF β1, tumornecrosis factor α (TNF-α), interleukin 1 (IL-1), IL-6, IL-8, COX-2, andmacrophage inflammatory protein (e.g., MIP-1α, and MIP-2).

In all of the above embodiments of the therapeutic methods of theinvention, the polypeptides of the invention can be used as the soleactive agent, or can be combined with one or more other treatments forthe indication, as determined by an attending physician.

As used herein for all of the methods of the invention, a“therapeutically effective amount” or an “amount effective” of the oneor more polypeptides is an amount that is sufficient to provide theintended benefit of treatment. An effective amount of the polypeptidesthat can be employed ranges generally between about 0.01 μg/kg bodyweight and about 10 mg/kg body weight, preferably ranging between about0.05 μg/kg and about 5 mg/kg body weight. However dosage levels arebased on a variety of factors, including the type of injury, the age,weight, sex, medical condition of the individual, the severity of thecondition, the route of administration, and the particular compoundemployed. Thus, the dosage regimen may vary widely, but can bedetermined routinely by a physician using standard methods.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describedthe methods and/or materials in connection with which the publicationsare cited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Experiments with Polypeptide WLRRIKAWLRRIKA-LNRQLGVAA (SEQ ID NO: 54,“BIP”) in an In vitro Model of Keloid Formation and HyperplasticScarring:

Keloid fibroblasts are an appropriate model of keloid formation andhyperplastic scarring. First, no reliable animal models of hyperplasticscarring exist. To our knowledge, no animal species provides a naturalnon-cancer related model of hyperplastic scarring. Furthermore, animalmodels of scarring that do exist use extreme methods to induceconsistent scarring such as full thickness burning of skin andallografting. [[D. Y. Yang, S. R. Li, G. Li, J. Y. Liu, Z. X. Wang, J.L. Wu, and Y. Q. Chen, “[Establishment of an animal model of humanhyperplastic scar in nude mice],” Zhonghua Shao Shang Za Zhi, vol. 20,pp. 82-4, April 2004.] These aggressive methods are much more extremethan the simple surgical cuts than often induce keloid formation orhyperplastic scarring in humans. Another investigator has used tissueengineered scaffolds to implant human keloid fibroblast in athyrnicmice. [H. B. Wang and S. K. Luo, “[Construction of animal models ofkeloid by tissue engineering],” Di Yi Jun Yi Da Xue Xue Bao, vol. 25,pp. 815-9, 832, July 2005.] Athymic, asplenic mice have also been usedto implant human keloid tissue into animals. [M. Polo, P. D. Smith, Y.J. Kim, X. Wang, F. Ko, and M. C. Robson, “Effect of TGF-beta2 onproliferative scar fibroblast cell kinetics,” Ann Plast Surg, vol. 43,pp. 185-90, August 1999.] However, in both of these cases, theinvestigators rely on human keloid fibroblasts as the primary cells intheir tissue implantations.

Second, in vitro keloid fibroblasts are widely used as models ofhyperplastic scarring by a myriad of other investigators interested instudying the biochemical basis of scarring and therapies to hyperplasticscarring. [R. J. Koch, R. L. Goode, and G. T. Simpson, “Serum-freekeloid fibroblast cell culture: an in vitro model for the study ofaberrant wound healing,” in Plast Reconstr Surg. vol. 99, 1997, pp.1094-8. W. Xia, M. T. Longaker, and G. P. Yang, “P38 MAP kinase mediatestransforming growth factor-beta2 transcription in human keloidfibroblasts,” Am J Physiol Regul Integr Comp Physiol, vol. 290, pp.R501-8, March 2006. W. Xia, W. Kong, Z. Wang, T. T. Phan, I. J. Lim, M.T. Longaker, and G. P. Yang, “Increased CCN2 transcription in keloidfibroblasts requires cooperativity between AP-1 and SMAD binding sites,”Ann Surg, vol. 246, pp. 886-95, November 2007. A. S. Vincent, T. T.Phan, A. Mukhopadhyay, H. Y. Lim, B. Halliwell, and K. P. Wong, “HumanSkin Keloid Fibroblasts Display Bioenergetics of Cancer Cells,” J InvestDermatol, Oct. 18, 2007. R. P. Abergel, D. Pizzurro, C. A. Meeker, G.Lask, L. Y. Matsuoka, R. R. Minor, M. L. Chu, and J. Uitto, “Biochemicalcomposition of the connective tissue in keloids and analysis of collagenmetabolism in keloid fibroblast cultures,” J Invest Dermatol, vol. 84,pp. 384-90, May 1985. K. Sahara, A. Kucukcelebi, F. Ko, L. Phillips, andM. Robson, “Suppression of in vitro proliferative scar fibroblastcontraction by interferon alfa-2b,” Wound Repair Regen, vol. 1, pp.22-7, January 1993. J. D. Russel, S. B. Russell, and K. M. Trupin, “Theeffect of histamine on the growth of cultured fibroblasts isolated fromnormal and keloid tissue,” J Cell Physiol, vol. 93, pp. 389-93, December1977. G. Pinol, F. Rueda, F. Marti, L. Puig, and J. M. De Moragas,“[Effect of minoxidil on DNA synthesis in cultured fibroblasts fromhealthy skin or keloids],” Med Cutan Ibero Lat Am, vol. 18, pp. 13-7,1990. M. C. McCormack, K. C. Nowak, and R. J. Koch, “The effect ofcopper tripeptide and tretinoin on growth factor production in aserum-free fibroblast model,” Arch Facial Plast Surg, vol. 3, pp. 28-32,January-March 2001. L. Y. Matsuoka, J. Uitto, J. Wortsman, R. P.Abergel, and J. Dietrich, “Ultrastructural characteristics of keloidfibroblasts,” Am J Dermatopathol, vol. 10, pp. 505-8, December 1988. J.Kossi and M. Laato, “Different metabolism of hexose sugars and sucrosein wound fluid and in fibroblast cultures derived from granulationtissue, hypertrophic scar and keloid,” Pathobiology, vol. 68, pp. 29-35,January-February 2000. R. H. Hong, J. Lum, and R. Koch, “Growth ofkeloid-producing fibroblasts in commercially available serum-freemedia,” Otolaryngol Head Neck Surg, vol. 121, pp. 469-73, October 1999.M. M. Hanasono, M. Kita, A. A. Mikulec, D. Lonergan, and R. J. Koch,“Autocrine growth factor production by fetal, keloid, and normal dermalfibroblasts,” Arch Facial Plast Surg, vol. 5, pp. 26-30,January-February 2003. A. Dalkowski, S. Fimmel, C. Beutler, and C.Zouboulis Ch, “Cryotherapy modifies synthetic activity anddifferentiation of keloidal fibroblasts in vitro,” Exp Dermatol, vol.12, pp. 673-81, October 2003. L. L. Chiu, C. H. Sun, A. T. Yeh, B.Torkian, A. Karamzadeh, B. Tromberg, and B. J. Wong, “Photodynamictherapy on keloid fibroblasts in tissue-engineeredkeratinocyte-fibroblast co-culture,” Lasers Surg Med, vol. 37, pp.231-44, September 2005. L. A. Carroll and R. J. Koch, “Heparinstimulates production of bFGF and TGF-beta 1 by human normal, keloid,and fetal dermal fibroblasts,” Med Sci Monit, vol. 9, pp. BR97-108,March 2003. L. A. Carroll, M. M. Hanasono, A. A. Mikulec, M. Kita, andR. J. Koch, “Triamcinolone stimulates bFGF production and inhibitsTGF-beta 1 production by human dermal fibroblasts,” Dermato Surg, vol.28, pp. 704-9, August 2002. M. Calderon, W. T. Lawrence, and A. J.Banes, “Increased proliferation in keloid fibroblasts wounded in vitro,”J Surg Res, vol. 61, pp. 343-7, March 1996. P. D. Butler, D. P. Ly, M.T. Longaker, and G. P. Yang, “Use of organotypic coculture to studykeloid biology,” Am J Surg, Dec. 5, 2007.] Keloid fibroblasts are linkedto increased extracellular matrix production. [R. P. Abergel, D.Pizzurro, C. A. Meeker, G. Lask, L. Y. Matsuoka, R. R. Minor, M. L. Chu,and J. Uitto, “Biochemical composition of the connective tissue inkeloids and analysis of collagen metabolism in keloid fibroblastcultures,” J Invest Dermatol, vol. 84, pp. 384-90, May 1985.] Inparticular, keloid cells are associated with increased type I collagenproduction, and type I collagen is the primary extracellular matrixmolecule found in hyperplastic scars and keloids. [R. P. Abergel, D.Pizzurro, C. A. Meeker, G. Lask, L. Y. Matsuoka, R. R. Minor, M. L. Chu,and J. Uitto, “Biochemical composition of the connective tissue inkeloids and analysis of collagen metabolism in keloid fibroblastcultures,” J Invest Dermatol, vol. 84, pp. 384-90, May 1985.] Clearly,keloid fibroblasts are an appropriate model for the study ofhyperplastic scarring and keloid formation.

Example 1 HSP27 Kinase Inhibitor Peptide (BIP) Inhibits TGF-β1 InducedExpression of CTGF and Collagen in Human Keloid Fibroblasts

Human keloid fibroblasts were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with fetal calf serum (10%) andstreptomycin/penicillin (1%). They were grown to 80% confluence, andthen serum starved for 48 hours. The cells were subsequently stimulatedwith nothing (control), with 1.25 ng/ml of transforming growth factorbeta 1 (TGF) for 24 hours, with 25 μM of a p38 MAP kinase inhibitor, SB203580, for 2 h followed by TGF-β1 (TGF+SB) or with 60 μM of testpolypeptide WLRRIKAWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54) for 2hours followed by TGF-β1 for 24 hours (TGF+BIP). The expression ofconnective tissue growth factor (CTGF) and collagen was assessed byimmunoblot and normalized to GAPDH (loading control) expression. Resultsin the graphs (FIG. 1) represent average±standard deviation from threeindependent experiments. These results demonstrate that BIP inhibitsTGF-β1 induced expression of CTGF and collagen in human keloidfibroblasts.

Example 2 HSP27 Kinase Inhibitor Peptide (BIP) Inhibits TGF-β1 InducedStress Fiber Formation in Human Keloid Fibroblasts

Human keloid fibroblasts were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with fetal calf serum (10%) andstreptomycin/penicillin (1%). They were grown on cover slips and thenserum starved for 48 hours. The cells were subsequently stimulated withnothing (control), with 1.25 ng/ml of transforming growth factor beta 1(TGF-β1) for 24 hours, or with 60 μM test polypeptideWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54) for 2 hours followed byTGF-β1 for 24 h (TGF-β1+BIP). Cells were washed, processed formicroscopy and labeled with Alexa 586-conjugated phalloidin to revealthe actin cytoskeleton and DAPI to reveal the nucleus. FIG. 2, at amagnification of 40×, demonstrates that pre-treatment of cells with BIPfollowed by TGF-β1 led to loss of central actin and reduced stress fiberformation in keloid fibroblasts.

Example 3 HSP27 Kinase Inhibitor Peptide (BIP) Inhibits TGF-β1 InducedPhosphorylation of HSP27 in Human Keloid Fibroblasts (FluorescenceMicroscopy)

Human keloid fibroblasts were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with fetal calf serum (10%) andstreptomycin/penicillin (1%). They were grown on cover slips and thenserum starved for 48 hours. The cells were subsequently stimulated withnothing (control), with 1.25 ng/ml of transforming growth factor beta 1(TGF-β1) for 24 hours or with 60 μM test polypeptideWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54) for 2 hours followed byTGF-β1 for 24 hours (TGF-β1+BIP). Cells were washed, processed formicroscopy and labeled with Cy2 for phospho-HSP27 (ser 78/82, greenfluorescence), Alexa 586-conjugated phalloidin (red) to reveal the actincytoskeleton and DAPI (blue) to reveal nucleus. FIG. 3, each panel shownat a magnification of 40×, shows that pre-treatment of cells with BIPfollowed by TGF-β1 reduced phosphorylation of HSP27 in keloidfibroblasts. These data demonstrated that BIP inhibits TGF-β1 inducedphosphorylation of HSP27 in human keloid fibroblasts.

Example 4 HSP27 Kinase Inhibitor Peptide (BIP) Inhibits TGF-β1 InducedPhosphorylation of HSP27 in Human Keloid Fibroblasts (Immunoblot)

Human keloid fibroblasts were maintained in Dulbecco's modified Eagle'smedium (DMEM) supplemented with fetal calf serum (10%) andstreptomycin/penicillin (1%). They were grown to 80% confluence, andthen serum starved for 48 hours. The cells were subsequently stimulatedwith nothing (control), with 1.5 ng/ml of transforming growth factorbeta 1 (TGF-β1) (shown as TGF in FIG. 4) for 24 hours (TGF), with 25 μMof a p38 MAP kinase inhibitor, SB 203580 for 2 hours followed by TGF-β1for 24 hours (TGF+SB), or with 60 μM test polypeptideWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54) for 2 hours followed byTGF-β1 for 24 hours (TGF+BIP). The phosphorylation of HSP27 was assessedby immunoblot analysis using antibodies against phospho-HSP27 (ser78/82), HSP27, and β actin as a loading control. These data are shown inFIG. 4A. The ratio of phospho-HSP27 to HSP27 was calculated, and thepercent phosphorylation was determined with respect to thephosphorylation obtained with TGF-β1 (TGF). These data are shown in FIG.4B. These data further demonstrate that BIP effectively inhibitedphosphorylation of HSP27.

Example 5 HSP27 Kinase Inhibitor Peptide (BIP) Inhibits Phosphorylationof HSP27 In Vitro

Recombinant HSP27 (1 μg) was phosphorylated with no enzyme (control,lane 1), MAPKAP kinase 2 (MAPKAPKII) (50 ng) in the absence (lane 2) orpresence of 200 μM KKKALNRQLGVAA (SEQ ID NO: 72) (published by Hayessand Benndorf) obtained from Calbiochem (lane 3), BIP (HSP27 kinaseinhibitor peptide, WLRRIKAWLRRIKA-LNRQLGVAA (SEQ ID NO:54) (lane 4), orHSP20 phosphopeptide (PTD-P20, lane 4) for 30 min at 30° C. The reactionmixtures were inactivated and separated by SDS-PAGE and transferred toPVDF membrane. The blot was probed with antibodies against HSP27 andphospho-HSP27. The ratio of phospho-HSP27 to HSP27 was calculated, andthe percent phosphorylation was determined with respect to thephosphorylation obtained in the absence of inhibitor (lane 2). The datais shown in FIG. 5. This blot is a representative of 2 separateexperiments. These data demonstrate that BIP inhibits MAPKAPKII inducedphosphorylation of HSP27 in vitro.

Experiments with Polypeptide WLRRIKAWLRRIKA-LNRQLGVAA (SEQ ID NO: 54,“BIP”) in an In vitro Model of Intimal Hyperplasia:

Example 6 The p38 MAP Kinase Inhibitor, SB 203580 and HSP27 KinaseInhibitor, BIP, Enhance Sodium Nitroprusside Induced Relaxation ofSaphenous Vein

Segments of human saphenous vein (HSV) were equilibrated in a musclebath and pretreated with buffer (control), 25 μM SB203580 (SB) or 30 μMWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54) for 2 hours and thencontracted with norepinephrine (NE, 0.5 μM) and relaxed with increasingdoses of sodium nitroprusside (SNP, 0-1 μM). The results are shown inFIG. 6. SB or BIP treatment led to significant increases in therelaxation of saphenous vein at 0.1 μM SNP compared to control.

Example 7 HSP27 Kinase Inhibitor does not Inhibit Endothelial CellProliferation

Human Aortic Endothelial Cells (Passage 2) (Cascade Biologics) wereseeded into 96-well plates at a concentration of 2000 cells per well asdetermined using a hemocytometer and allowed to adhere for 4 hours. Theywere then treated with a final concentration of 1, 10 or 50 μMWLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO: 54), 1, 10, or 50 μMscrambled peptide (SCR), or an equal volume of phosphate buffered saline(PBS). Cells were returned to the incubator for 16 hours and counted ona Molecular Devices M5 spectrophotometer after 1 hour treatment withCyQuant NF Cell Proliferation Assay (Invitrogen). Results of theexperiment are shown in FIG. 7. These data show that at doses where thepolypeptides of the invention suppress CTGF and collagen deposition byfibroblasts, they do not inhibit endothelial cell proliferation,supporting the hypothesis that the polypeptides will be effective atinhibiting intimal hyperplasia and not inhibiting endothelial celllining if delivered from grafts and stents.

Conclusion

Examples 1-7 demonstrate that WLRRIKAWLRRIKA-LNRQLGVAA (BIP) (SEQ ID NO:54) inhibits TGF-β1 induced expression of CTGF and collagen in humankeloid fibroblasts, leads to loss of central actin and reduced stressfiber formation in keloid fibroblasts, inhibits TGF-β1 inducedphosphorylation of HSP27 in human keloid fibroblasts, inhibits MAPKAPkinase 2 induced phosphorylation of HSP27 in vitro, and increases therelaxation of saphenous vein. The examples further demonstrate that BIPdoses where the polypeptide suppresses CTGF and collagen deposition byfibroblasts do not inhibit endothelial cell proliferation, supportingthe hypothesis that the polypeptides will be effective at inhibitingintimal hyperplasia and not inhibiting endothelial cell lining ifdelivered from grafts and stents.

Example 8 Alanine Scanning Mutagenesis

Amino acids 3-9 of the peptide KALNRQLGVAA (SEQ ID NO:60) weresequentially replaced with alanine, resulting in the following peptides:

KAANRQLGVAA (SEQ ID NO: 63) KALARQLGVAA (SEQ ID NO: 64) KALNAQLGVAA (SEQID NO: 65) KALNRALGVAA (SEQ ID NO: 66) KALNRQAGVAA (SEQ ID NO: 67)KALNRQLAVAA (SEQ ID NO: 68) KALNRQLGAAA (SEQ ID NO: 69)

These peptides were tested in MAPKAP-K2 kinase inhibition assays andcompared to the peptides KALNRQLGVAA (SEQ ID NO: 60), KKKALNRQLGVAA (SEQID NO: 72) (Calbiochem), or negative controls.

Briefly, a MAPKAP kinase II assay kit from Invitrogen was used for allassays. Cocktails containing varying inhibitor concentrations, asubstrate peptide and kinase were prepared and pipetted into 96-wellplates. The plates were placed in an M5 (Molecular Devices) andmaintained at 30 C. Readings were taken every 20 seconds for 20 minutesand the resulting reaction velocities were obtained. A summary of thedate is presented in Table 1.

TABLE 1 Reaction Velocities for MK2 Inhibitor Variants (n = 3) % ofKALNRQLGVAA Reaction Velocity at an Inhibitor Concentration of PeptideSequence SEQ ID NO: 100 μM (+/−SEM*) KALNRQLGVAA 62 100% (+/−3%)KALNRQLGVA 70 100% (+/−3%) KAANRQLGVAA 63 152% (+/−3%) KALARQLGVAA 64 39% (+/−1%) KALNAQLGVAA 65 358% (+/−8%) KALNRALGVAA 66  358% (+/−15%)KALNRQAGVAA 67 118% (+/−4%) KALNRQLAVAA 68  72% (+/−3%) KALNRQLGAAA 69 373% (+/−13%) KAdLNRQLGVAA 71 146% (+/−4%) KALdNRQLGVAA 72  95% (+/−6%)KALNdRQLGVAA 73 306% (+/−4%) KALNRdQLGVAA 74 276% (+/−3%) KALNRQdLGVAA75  357% (+/−10%) KALNRQLGdVAA 76  260% (+/−14%) KKKALNRQLGVAACommercially available  91% (+/−4%) from Calbiochem *SEM = StandardError of the Mean for three values

All peptides were used at the same concentration. The results of theseexperiments demonstrated the following in comparison to KALNRQLGVAA (SEQID NO: 60).

KAANRQLGVAA (SEQ ID NO: 61): Retains inhibitory activity, but reduced byup to approximately 30%;

KALARQLGVAA (SEQ ID NO: 62): Activity increased by approximately 60%compared to KALNRQLGVAA (SEQ ID NO: 60);

KALNAQLGVAA (SEQ ID NO: 63): Eliminates almost all inhibitory activity;

KALNRALGVAA (SEQ ID NO:64): Eliminates most inhibitory activity;

KALNRQAGVAA (SEQ ID NO:65): Retains inhibitory activity, but reduced byup to approximately 25%;

KALNRQLAVAA (SEQ ID NO: 66): Activity increased by approximately 25%compared to KALNRQLGVAA (SEQ ID NO:60);

KALNRQLGAAA (SEQ ID NO: 67): Eliminates almost all inhibitory activity.

These data indicate that residues 5 (R), 6 (Q), and 9 (V) play a keyrole in peptide activity.

Experimental results have demonstrated that the C-terminal alanine isnot necessary for activity. Alanine substitution at residues 3 and 7 haslittle effect on inhibitory activity, and thus it is believed that theseresidues can be substituted with amino acids of similar size. Alaninesubstitutions at residues 4 and 8 increase activity compared toKALNRQLGVAA (SEQ ID NO: 60), suggesting the substitutions at thesepositions with small amino acids may increase activity.

Alanine substitutions at residues 5, 6, and 9 have strong effects oninhibitory activity, and thus it is likely that these positions favorvery similar amino acids (i.e., size and charge profile) to thosepresent in the starting polypeptide.

HSP27 Kinase Inhibitor Peptides Inhibit LPS-Induced TNF-α Excretion inan In Vitro Model of Inflammation.

HSP27 kinase inhibitor peptides KAF and FAK inhibit LPS-induced TNF-αexcretion of THP-1 monocytes. The expression and regulation of cytokinesis central to the onset and progression of many disease states includinginflammatory disorders and diseases. [Brennan, F. M.; Maini, R. N.;Feldmann, M., Cytokine Expression in Chronic Inflammatory Disease.British Medical Bulletin 1995, 51, (2), 368-384.] Although not anexhaustive list, these disease states include rheumatoid arthritis,chronic obstructive pulmonary disease, atherosclerosis, asthma, Crohn'sdisease, inflammatory bowel disease, osteoarthritis, tendonitis, andpsoriasis. One example of a well-studied disease involving cytokineoverexpression is rheumatoid arthritis. Rheumatoid arthritis ischaracterized by chronic inflammation resulting in debilitatingconsequences ranging from pain and disability to premature mortality.[Jenkins, J. K.; Hardy, K. J.; McMurray, R. W., The pathogenesis ofrheumatoid arthritis: A guide to therapy. American Journal of theMedical Sciences 2002, 323, (4), 171-180. Pincus, T.; Callahan, L. F.,What Is the Natural-History of Rheumatoid-Arthritis. Rheumatic DiseaseClinics of North America 1993, 19, (1), 123-151. Although the etiologyof rheumatoid arthritis remains unknown, the chronic nature of thedisease seems to be perpetuated by cytokines and other factors secretedby activated leukocytes recruited to the synovium. [Jenkins, J. K.;Hardy, K. J.; McMurray, R. W., The pathogenesis of rheumatoid arthritis:A guide to therapy. American Journal of the Medical Sciences 2002, 323,(4), 171-180. Firestein, G. S.; Zvaifler, N.J., How important are Tcells in chronic rheumatoid synovitis?II. T cell-independent mechanismsfrom beginning to end. Arthritis and Rheumatism 2002, 46, (2), 298-308.]Within the synovial joint of patients with rheumatoid arthritis,cytokines including tumor necrosis factor-α (TNF-α), interleukin 1β(IL-1β), IL-6, IL-8, and granulocyte macrophage colony stimulatingfactor (GM-CSF) have been identified at elevated levels relative tothose found in healthy synovial joints. In particular, TNF-α and IL-1βhave been implicated as major mediators of inflammation in rheumatoidarthritis, and both greatly influence the expression of otherproinflammatory cytokines. [Firestein, G. S.; Zvaifler, N.J., Howimportant are T cells in chronic rheumatoid synovitis?II. Tcell-independent mechanisms from beginning to end. Arthritis andRheumatism 2002, 46, (2), 298-308. Feldmann, M.; Brennan, F. M.; Maini,R. N., Role of cytokines in rheumatoid arthritis. Annual Review ofImmunology 1996, 14, 397-440. Feldmann, M.; Maini, R. N., The role ofcytokines in the pathogenesis of rheumatoid arthritis. Rheumatology1999, 38, 3-7.] Produced principally by activated macrophages in thesynovial membrane, TNF-α and IL-1β not only stimulate expression of eachother but also stimulate fibroblast-like synoviocytes to synthesize andsecrete a variety of factors that further progress rheumatic disease.[Jenkins, J. K.; Hardy, K. J.; McMurray, R. W., The pathogenesis ofrheumatoid arthritis: A guide to therapy. American Journal of theMedical Sciences 2002, 323, (4), 171-180.] During the past decade, asignificant body of literature has described more of the intracellularsignal transduction pathways implicated in rheumatoid arthritis. Many ofthese studies have implicated mitogen-activated protein kinases (MAPKs)as enzymes important in the synthesis of inflammatory mediators of manydiseases including rheumatoid arthritis. [Saklatvala, J., The p38 MAPkinase pathway as a therapeutic target in inflammatory disease. CurrentOpinion in Pharmacology 2004, 4, (4), 372-377.] In particular,inhibitors of p38 MAP kinase and its downstream target MAPK-activatedprotein kinase 2 (MAPKAP kinase 2) have been shown to downregulateproduction of cytokines such as TNF-α and IL-1β. [Kumar, S.; Boehm, J.;Lee, J. C., p38 map kinases: Key signaling molecules as therapeutictargets for inflammatory diseases. Nature Reviews Drug Discovery 2003,2, (9), 717-726.] Although newer approaches in rheumatoid arthritis drugdesign have focused on small molecule inhibitors of p38 MAP kinase andto a lesser extent MAPKAP kinase 2, little attention has been given tothe development of biologic therapeutics targeting MAPKAP kinase 2.[Gaestel, M.; Mengel, A.; Bothe, U.; Asadullah, K., Protein kinases assmall molecule inhibitor targets in inflammation. Current MedicinalChemistry 2007, 14, (21), 2214-2234.] Although many small molecularinhibitors of p38 MAP kinase have reduced TNF-α production in THP-1 celllines and decreased observed characteristics/symptoms of inflammatory inanimal models, p38 MAP kinase knockout mice are not viable, and many ofthe small molecules have shown side effects including liver toxicity.Since MAPKAP kinase 2 is a substrate of p38 MAP kinase and becauseMAPKAP kinase 2 knockout mice are viable and have shown decreasedproduction of TNF-α and IL-6, a more selective inhibitor of MAPKAPkinase 2 (also referred to as HSP27 kinase) likely could providespecific inhibition with a decrease in side effects. [Hegen, M.;Gaestel, M.; Nickerson-Nutter, C. L.; Lin, L. L.; Telliez, J. B., MAPKAPkinase 2-deficient mice are resistant to collagen-induced arthritis.Journal of Immunology 2006, 177, (3), 1913-1917.] Although the mechanismof action is still unknown, one possibility is that HSP27, a substrateof MAPKAP kinase 2 that can be phosphorylated on serines 15, 78 or 82(in human) acts to stabilize transcription factors or mRNA related toproinflammatory cytokines synthesis. Also, MAPKAP kinase 2 has beenshown to stabilize complexes with p38 involved in maintaining stabilityof proinflammatory mRNA. [Saklatvala, J., The p38 MAP kinase pathway asa therapeutic target in inflammatory disease. Current Opinion inPharmacology 2004, 4, (4), 372-377. Kumar, S.; Boehm, J.; Lee, J. C.,p38 map kinases: Key signaling molecules as therapeutic targets forinflammatory diseases. Nature Reviews Drug Discovery 2003, 2, (9),717-726.] Recent work has implicated MAPKAP kinase 2 as an essentialprotein for lipopolysaccharide (LPS) induced TNF-α synthesis.[Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert, R; Birchmeier, C.;Volk, H. D.; Gaestel, M., MAPKAP kinase 2 is essential for LPS-inducedTNF-alpha biosynthesis. Nature Cell Biology 1999, 1, (2), 94-97.]

THP-1 monocytes are used routinely as a model cell line for induction ofcytokines upon stimulation with lipopolysaccharide (LPS). Cytokineexcretion can be enhanced further when the cells are firstdifferentiated with phorbol 12-myristate 13-acetate (PMA) intomacrophage-like cells. [Auwerx, J., The Human Leukemia-Cell Line,Thp-1-a Multifaceted Model for the Study of Monocyte-MacrophageDifferentiation. Experientia 1991, 47, (1), 22-31.] THP-1 monocytes andLPS-induced THP-1 monocytes have been used as a model to study efficacyof antimicrobial agents, [Takemura, H.; Yamamoto, H.; Kunishima, H.;Ikejima, H.; Hara, T.; Kanemitsu, K.; Terakubo, S.; Shoji, Y.; Kaku, M.;Shimada, J., Evaluation of a human monocytic cell line THP-1 model forassay of the intracellular activities of antimicrobial agents againstLegionella pneumophila. Journal of Antimicrobial Chemotherapy 2000, 46,(4), 589-594] general toxicity of compounds, [Sestier, C.; Lacava, Z. G.M.; Lacava, L. M.; Da Silva, M. F.; Azevedo, R. B.; Buske, N.; Gansau,C.; Morais, P. C.; Silva, O.; Pelegrini, F.; Sabolovic, D., In vitrotoxicity of magnetic fluids evaluated for macrophage cell lines. Journalof Magnetism and Magnetic Materials 2002, 252, (1-3), 403-405.], andespecially as models of activated macrophages. [Auwerx, J., The HumanLeukemia-Cell Line, Thp-1-a Multifaceted Model for the Study ofMonocyte-Macrophage Differentiation. Experientia 1991, 47, (1), 22-31.]With regard to applications involving inflammation, THP-1 cells haveoften been used as a model system to investigate efficacy ofanti-inflammatory agents including p38 kinase inhibitors. [Ross, S.;Chen, T.; Yu, V.; Tudor, Y.; Zhang, D. W.; Liu, L. B.; Tamayo, N.;Dominguez, C.; Powers, D., High-content screening analysis of the p38pathway: Profiling of structurally related p38 alpha kinase inhibitorsusing cell-based assays. Assay and Drug Development Technologies 2006,4, (4), 397-409.] Often, excreted cytokine levels are compared betweencontrols and treatments containing anti-inflammatory agents to determinedrug efficacy.

Phorbol 12-myristate 13-acetate (“PMA”) is a potent tumor promote thatactivates the signal transduction enzyme protein kinase C (“PKC”). PKCphosphorylates proteins altering their function. Its effects arecell-specific.

Example 9 HSP27 Kinase Inhibitor Peptide KAF and FAK Inhibit LPS-InducedTNF-α Excretion in an In Vitro Model of Inflammation

Human THP-1 monocytes were maintained in RPMI 1640 supplemented withfetal bovine serum (10%), streptomycin/penicillin (1%), 0.05 mMβ-mercaptoethanol, 1 mM sodium pyruvate, and 10 mM HEPES. They wereseeded at a density of 250,000 cells/ml and treated with 200 nM phorbol12-myristate 13-acetate (PMA) for 20 hours. One cohort was not treatedwith PMA as a control (cells). Then, cells were treated with one of thefollowing treatments: no treatment and no PMA (cells), no treatment(PMA), 100 ng/ml lipopolysaccharide (LPS) (PMA, LPS), 100 ng/ml LPS with10 μM SB203580 (SB), or 100 ng/ml LPS with various concentrations ofHSP27 kinase inhibitor peptides. The inhibitor peptides wereKAFAKLAARLYRKALARQLGVAA (KAF) (SEQ ID NO: 48) and FAKLAARLYRKALARQLGVAA(FAK) (SEQ ID NO: 49), and the treatment concentrations were 30 μM (KAF30 uM or FAK 30 uM), 10 μM (KAF 10 uM or FAK 10 uM), 3 μM (KAF 3 uM orFAK 3 uM), and 1 μM (KAF 1 uM or FAK 1 uM). After applying thetreatments, the cells were grown for 6 hours at 37° C. and 5% CO2. Then,the cells were centrifuged, and TNF-α concentration in the supernatantwas determined by ELISA. Viability of centrifuged cells was determinedusing an MTT-based assay. Results of cellular viability presented inFIG. 8 represent average±standard deviation of four replicates. Resultsof relative TNF-α excretion presented in FIG. 9 representaverage±standard deviation of four replicates. These results demonstratethat KAF and FAK inhibit LPS-induced TNF-α excretion from monocytes in adose dependent manner without adversely affecting cellular viability.

Example 10 HSP27 Kinase Inhibitor Peptides BIP and YARA (SEQ ID NO: 73)Inhibit LPS-Induced TNF-α Excretion of THP-1 Monocytes

Human THP-1 monocytes were maintained in RPMI 1640 supplemented withfetal bovine serum (10%), streptomycin/penicillin (1%), 0.05 mMβ-mercaptoethanol, 1 mM sodium pyruvate, and 10 mM HEPES. They wereseeded at a density of 250,000 cells/ml and treated with 200 nM phorbol12-myristate 13-acetate (PMA) for 20 hours. One cohort was not treatedwith PMA as a control (cells). Then, cells were treated with one of thefollowing treatments: no treatment and no PMA (cells), no treatment(PMA), 100 ng/ml lipopolysaccharide (LPS) (PMA, LPS), 100 ng/ml LPS with300 μM, 30 μM, 10 μM, or 1 μM SB203580 (SB), or 100 ng/ml LPS withvarious concentrations of HSP27 kinase inhibitor peptides. The inhibitorpeptides were WLRRIKAWLRRIKALNRQLGVAA (BIP) (SEQ ID NO: 54) andYARAAARQARAKALNRQLGVA (YARA (“YARA”disclosed as SEQ ID NO: 73) (SEQ IDNO: 55). For BIP, the treatment concentrations were 30 μM, 10 μM, 3 μM,1 μM, 100 nM, and 10 nM. For YARA (SEQ ID NO: 73), the treatmentconcentration was 1 mM. After applying the treatments, the cells weregrown for 6 hours at 37° C. and 5% CO02. Then, the cells werecentrifuged, and TNF-α concentration in the supernatant was determinedby ELISA. Viability of centrifuged cells was determined using anMTT-based assay. Results of cellular viability presented in FIG. 10represent average±standard deviation of four replicates. Results ofrelative TNF-α excretion presented in FIG. 11 represent average±standarddeviation of four replicates. These results demonstrate that BIP andYARA (SEQ ID NO: 73) can be used at concentrations which inhibitLPS-induced TNF-α excretion from monocytes without adversely affectingcellular viability.

Experiments in an In Vitro Model for Gliosis and Scar Formation in theCentral Nervous System

Primary astrocyte cultures are an appropriate in vitro model for gliosisand scar formation in the central nervous system. Astrocytes are theprimary cell type implicated in gliosis and scar formation in thecentral nervous system. Gliosis and scar formation are believed to betriggered by cytokines including TGF-β1, IL6 and TNF-α and serum derivedfactors including LPA and endothelins. Their activity is propagated bycyclic nucleotide-dependent signaling resulting in p38 MAP kinaseactivation followed by MAPKAP kinase II (MK2) activation. Yang, et al.demonstrated that three cytokines, IL-1β, IL-6 and TNF-α play roles bothin spinal cord tissue regeneration and in tissue damage. They furthersuggest that low levels of expression are likely to be beneficial totissue maintenance and regeneration, but higher levels of expression aredetrimental [Yang, L. L., et al., Early expression and cellularlocalization of proinflammatory cytokines interleukin-1 beta,interleukin-6, and tumor necrosis factor-alpha in human traumatic spinalcord injury. Spine, 2004. 29(9): p. 966-71.] Multiple studies suggestthat over expression of these cytokines leads to exacerbated secondaryinjury [Yang, L. L., et al., Early expression and cellular localizationof proinflammatory cytokines interleukin-1beta, interleukin-6, and tumornecrosis factor-alpha in human traumatic spinal cord injury. Spine,2004. 29(9): p. 966-71. Velardo, M. J., et al., Patterns of GeneExpression Reveal a Temporally Orchestrated Wound Healing Response inthe Injured Spinal Cord. J. Neurosci., 2004. 24(39): p. 8562-8576.Bareyre, F. M. and M. E. Schwab, Inflammation, degeneration andregeneration in the injured spinal cord: insights from DNA microarrays.Trends Neurosci, 2003. 26(10): p. 555-63. Pineau, I. and S. Lacroix,Proinflammatory cytokine synthesis in the injured mouse spinal cord:multiphasic expression pattern and identification of the cell typesinvolved. J Comp Neurol, 2007. 500(2): p. 267-85.] [Yang, L. L., et al.,Early expression and cellular localization of proinflammatory cytokinesinterleukin-1beta, interleukin-6, and tumor necrosis factor-alpha inhuman traumatic spinal cord injury. Spine, 2004. 29(9): p. 966-71.Velardo, M. J., et al., Patterns of Gene Expression Reveal a TemporallyOrchestrated Wound Healing Response in the Injured Spinal Cord. J.Neurosci., 2004. 24(39): p. 8562-8576. Bareyre, F. M. and M. E. Schwab,Inflammation, degeneration and regeneration in the injured spinal cord:insights from DNA microarrays. Trends Neurosci, 2003. 26(10): p. 555-63.Pineau, I. and S. Lacroix, Proinflammatory cytokine synthesis in theinjured mouse spinal cord: multiphasic expression pattern andidentification of the cell types involved. J Comp Neurol, 2007. 500(2):p. 267-85.], while, as discussed above, complete depletion of cytokinesignaling can lead to a decrease in neuronal survival [Iwasaki, Y., etal., Effect of transforming growth factor beta 1 on spinal motor neuronsafter axotomy. J Neurol Sci, 1997. 147(1): p. 9-12. Silver, J. and J. H.Miller, Regeneration beyond the glial scar. Nature Reviews Neuroscience,2004. 5(2): p. 146-156.]. The approach proposed herein inhibits MK2 andthus down-regulates expression of inflammatory cytokines due totraumatic brain injury and spinal cord injury.

Example 11 HSP27 Kinase Inhibitor Peptide KAF Decreases Phosphorylationof HSP27 in Astrocytes Even in the Presence of TNF-α

Primary rat astrocytes were isolated from cortical E19 rat brain tissue(BrainBits, LLC) following the protocol suggested by Brain Bits. Cellswere resuspended in Neurobasal media containing 10% serum and 3 mMglutamine and plated at a density of 75,000 cells/cm² in 10-cm Petridishes. Once confluent, cells were passaged twice with a 3:1 split priorto treatment. Cells were then treated with 5 ng/ml TNF-α to activatethem. Negative controls were untreated, positive controls treated withTNF-α alone, and two dishes were treated with KAF (SEQ ID NO:48) at 10μM or 30 μM. 24 hours after treatment, media was removed, cells washedwith PBS and lysed. Lysates were run on SDS-PAGE followed by transfer toPVDF membrane and western blot probing for phosphorylated HSP27. GAPDHwas used as a loading control. The results of this experiment are shownin FIG. 12. These results demonstrate that KAF (SEQ ID NO: 48) decreasesphosphorylation of HSP27 in astrocytes even when the astrocytes arechallenged with TNF-α.

Animal Studies

In another aspect, the present invention further describes experimentsin animal models of human disease that will be used to determine theeffect of the polypeptides of the present invention. These animal modelshave been used by other investigators, and are generally accepted assuch. The therapeutic results obtained with this model therefore can beextrapolated to methods of treating human subjects.

Animal Models

Intestinal adhesions model. Animal Studies will be carried out in theAAALAC accredited animal facilities at Purdue University in accordancewith the National Institutes of Health Guide for Care and Use ofAnimals. Male Sprague-Dawley rat weighing between 240-280 g will beincluded in the study. The cohorts have been designed to include apositive control, cecum abrasion, no treatment, and a negative control,no abrasion, no treatment, as well as additional cohorts to evaluate theoptimal delivery method to prevent intestinal adhesions. All animalswill be maintained in separate cages under a 12 hour light/dark cycleand provided food and water ad libitum both before and followingsurgery. All animals will be anesthetized using an intra peritonealinjection of ketamine (75-100 mg/kg) and xylazine (5-10 mg/kg).Anesthesia will be maintained with an intra peritoneal injection of 10%induction dose of ketamine/xylazine. Anesthetic levels will be assessedusing the toe pinch method. Also, the animal's respiration and color ofmucous membrane will be monitored during the procedure. Animals will beeuthanized using barbiturate overdose (e.g., Nembutal 120 mg/kg) orsimilar commercially available euthanasia solution at the recommendeddosage IV or IP.

Anesthetized rats will be prepped for surgery by shaving the lowerabdomen and cleaning it with iodine. Animals will undergo a midlineceliotomy, the cecum will be identified and placed onto a gauze pad andsaline used to keep the tissue moist. The cecum wall will be abradedusing 1×1 cm electrosurgical tip cleaner, Johnson and Johnson, untilbleeding is noted on the anterior surface. A 1.6×0.8 mm defect will becreated in the peritoneum and underlying muscle using a 0.8 mm biopsypunch. The abdominal cavity will be irrigated prior to application oftreatments. The appropriate treatment will be applied between thejuxtaposed cecum and injured peritoneum. Specifically, in cohort 1 theabraded cecum will be juxtapose to the injured peritoneum and thesurgical incision closed. Cohort 2 be subjected to only the celiotomyand the incision will be closed. Additional cohorts will be irrigatedwith 10 mls of PBS containing the appropriate concentration, of MK2inhibitor If injury such as a perforated bowel occurs during surgery orthe barrier fails to separate the damaged tissue, the animal will beremoved from the study and replaced [Buckenmaier, C.C., 3rd, et al.,Comparison of antiadhesive treatments using an objective rat model. AmSurg, 1999. 65(3): p. 274-82; Zong, X., et al., Prevention ofpostsurgery-induced abdominal adhesions by electrospun bioabsorbablenanofibrous poly(lactide-co-glycolide)-based membranes. Ann Surg, 2004.240(5): p. 910-5].

Fourteen days post-surgery the rats will again be anesthetized asdescribed above and a surgeon who is blinded to the treatments willperform a second celiotomy to evaluate the extent and severity of theadhesions. The vast majority of abdominal adhesion studies use a visualanalogue scoring system rather than histology. The following scoringsystem will be used: 0=no adhesions, 1=thin and filmy, easily separatedadhesions, 2=significant and filmy, difficult to separate tissue and3=severe with fibrosis, instruments required to separate tissue. Thenumber of animals within each group with adhesions and the severity ofadhesions will be noted and then compared across groups using ANOVAanalysis to determine the best treatment combination (barrier, rate ofrelease and drug concentration) to inhibit adhesions.

Wound Healing/Scar Inhibition

Rat model of dermal scarring. A Sprague-Dawley model of scarring will beused. All protocols will have Institutional Animal Care and UseCommittee approval. A mixture of male and female animals will between240-280 g will be used. The animals will be anesthetized using mouseketamine cocktail (2 ml/kg, 21 mg/ml ketamine, 2.4 mg/ml xylazine, and0.03 mg/ml acepromazine). The area on the back will be shaved withsurgical clippers and scrubbed with Chlorhexidine surgical scrub. Asingle linear incision 2 cm in length extending through the dermis,epidermis, and into the subcutaneous fat will be made on the upper back.The incision will be closed with interrupted 4-0 nylon sutures.Treatment with 0.1 ml of increasing concentrations from 0.01 mM to 1 mMMK2i or saline vehicle will be subcutaneously injected on each side ofthe incision immediately after closure (n=7-8 animals per group).Control groups will have incisions, but no peptide treatment. Sutureswill be removed after 1 week, and animals were euthanized at 7, 14, and21 days post surgery by inhalation of carbon dioxide. At termination,the healing wounds and adjacent skin (as a control) will be excised. Thetissue will be placed in formalin, embedded in paraffin, and processedfor histologic sectioning and Masson's trichrome staining. Eachtrichrome-stained specimen will be blindly scored in duplicate by apathologist for dermal collagen fiber orientation, density, andmaturity.

Arthritis Models

Accepted in vivo animal models for rheumatoid arthritis includecollagen-induced arthritis (CIA), rat carrageenin-induced acute model ofinflammation, adjuvant-induced arthritis in rats (AA), and streptococcalcell wall-induced arthritis. Each of these models have been used to testcompounds inhibiting expression, excretion, and/or activity ofinflammatory cytokines. However, CIA is the murine model most commonlyused.

For collagen-induced arthritis (CIA), mice (6-8 weeks old) will beinjected in the base of the tail with 0.1 ml of a mixture of bovine typeII and complete Freund's adjuvant containing heat-killed Mycobacteriumtuberculosis. After 21 days, a 0.1 ml of collagen type II in phosphatebuffered saline (PBS) will be given as an injection to the base of thetail as a booster. (Hegen, M.; Gaestel, M.; Nickerson-Nutter, C. L.;Lin, L. L.; Telliez, J. B., MAPKAP kinase 2-deficient mice are resistantto collagen-induced arthritis. Journal of Immunology 2006, 177, (3),1913-1917; Yamanishi, Y.; Boyle, D. L.; Pinkoski, M. J.; Mahboubi, A.;Lin, T.; Han, Z. N.; Zvaifler, N.J.; Green, D. R.; Firestein, G. S.,Regulation of joint destruction and inflammation by p53 incollagen-induced arthritis. American Journal of Pathology 2002, 160,(1), 123-130; Podolin, P. L.; Callahan, J. F.; Bolognese, B. J.; Li, Y.H.; Carlson, K.; Davis, T. G.; Mellor, G. W.; Evans, C.; Roshak, A. K.,Attenuation of murine collagen-induced arthritis by a novel, potent,selective small molecule inhibitor of I kappa B kinase 2, TPCA-1(2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide),occurs via reduction of proinflammatory cytokines and antigen-induced Tcell proliferation. Journal of Pharmacology and ExperimentalTherapeutics 2005, 312, (1), 373-381). HSP27 kinase inhibitor in PBSwill be injected into the synovial joint of the hind limb. Controlanimals will receive an injection of PBS without inhibitor. Injectionswill begin once an animal has obtained a clinical score greater or equalto “1” for two consecutive days. Assessment of the arthritic state ofthe mice will be assessed using clinical arthritis scores of 0 (noarthritis), 1 (ankle swelling), 2 (ankle and midfoot swelling), 3(ankle, midfoot, and metatarsal joint swelling), and 4 (ankle, midfoot,metatarsal joint, and digit swelling). (Yamanishi, Y.; Boyle, D. L.;Pinkoski, M. J.; Mahboubi, A.; Lin, T.; Han, Z. N.; Zvaifler, N.J.;Green, D. R.; Firestein, G. S., Regulation of joint destruction andinflammation by p53 in collagen-induced arthritis. American Journal ofPathology 2002, 160, (1), 123-130; Podolin, P. L.; Callahan, J. F.;Bolognese, B. J.; Li, Y. H.; Carlson, K.; Davis, T. G.; Mellor, G. W.;Evans, C.; Roshak, A. K., Attenuation of murine collagen-inducedarthritis by a novel, potent, selective small molecule inhibitor of Ikappa B kinase 2, TPCA-1(2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide),occurs via reduction of proinflammatory cytokines and antigen-induced Tcell proliferation. Journal of Pharmacology and ExperimentalTherapeutics 2005, 312, (1), 373-381). Scoring of the contralateral hindlimb will also be performed. Samples of synovial fluid will be takenregularly for analysis of cytokine concentrations. After sacrificing themice, hind limbs will be fixed in formalin and processed for histologyto examine inflammation and hyperplasia. (Yamanishi, Y.; Boyle, D. L.;Pinkoski, M. J.; Mahboubi, A.; Lin, T.; Han, Z. N.; Zvaifler, N.J.;Green, D. R.; Firestein, G. S., Regulation of joint destruction andinflammation by p53 in collagen-induced arthritis. American Journal ofPathology 2002, 160, (1), 123-130.)

Spinal Cord Models

Spinal cord experiments: Sprague Dawley rats (200-300 gm) will besubjected to spinal cord injury using transection (Widenfalk,Lundstromer et al. 2001). Following halothane anesthesia, dorsallaminectomies at T9 expose the cord. Complete transection leaving a 2 mmgap will be achieved using an iris scalpel. Peptide concentrationsranging from 0.01-1 mM peptide or saline (control) will be applied tothe injured cord is achieved using 50-100 μl volumes. Closure will bedone using absorbable suture material, and the animals recover on warmedblankets. Prophylactic antibiotics will be administered for one week,and subsequently if needed. Urinary bladders will be emptied thricedaily by mechanical expression for the first week, and twice dailythereafter to prevent urinary tract infections. Animals will besacrificed at two time points to provide assessment of the onset andsustained regeneration of axons (typically in cohorts of 6 and 16 ondays 7 and 56 respectively. The day 7 time allows determination of theextent of proliferation of astrocytes and if there is a chronic immuneresponse. Day 56 will provide information on axonal regeneration(Coumans, J. V., T. T.-S. Lin, et al. (2001). “Axonal regeneration andfunctional recovery after complete spinal cord transection in rats bydelayed treatment with transplants and neurotrophins.” The Journal ofNeuroscience 21(23): 9334-9344). A larger number of animals is neededfor day 56 animals so that longitudinal and axonal sectioning as well asneuroanatomical tracing can be done (Woerly, Doan Woerly, S., V. D.Doan, et al. (2001). “Spinal cord reconstruction using Neurogel™Implants and functional recovery after chronic injury.” Journal ofNeuroscience Research 66: 1187-1197).

Spinal Cord Histology: Animals will be euthanized by CO2 inhalationaccording to AVMA recommendations (Andrews, E. J., B. T. Bennett, et al.(1993). “Report of the AVMA panel on Euthanasia.” Journal of theAmerican Veterinary Association 202(2): 229-249). Cardiac perfusionusing 2% paraformaldehyde in PBS, followed by 10% sucrose precedes corddissection to optimize histology (Andrew, D. and A. D. Craig,Spinothalamic lamina I neurons selectively sensitive to histamine: acentral neural pathway for itch. Nature Neuroscience, 2001. 4(1): p.72-77.). From the four animals not receiving neural tracing, the cord inthe region of injury will be recovered, then processed by longitudinalcryostat sectioning (14 μm) along the injured axis. For assessment ofproliferative cells in the injury site, anti-PCNA antibodies are appliedaccording to supplier's instruction. Cell-type staining for occupationof the matrix in the context of spinal repair will include astrocytes(glial fibrillary acidic protein, GFAP), oligodendrocytes (myelinproteolipid protein, mPLP), neurons (neuron specific enolase, NSE),GAP-43 (found in the growth cone of extending axons),monocytes/macrophages (CD45), lymphocytes (CD16), and endothelial cells(factor VIII).

Cervical contusion injury. A contusion injury will be created using anelectromagnetic SCI device. Animals will first be anesthetized and thena vertical incision will be made along the cervical vertebra and thesuperficial muscle and skin retracted. A laminectomy will be used toexpose at cervical vertebra C5 and the spinal cord underneath (C5) whilemaintaining an intact dura mater. The cervical contusion injury will becreated with a force of 3 Kdyn. The exposed C5 spinal cord will be ratedas either mildly, moderately or severely injured as determined bydisplacement of the spinal cord by 0.80, 0.95, or 1.10 mm, respectively,with a single, brief displacement of 20 msec. After injury, the musclesand skin will be sutured in layers. The rats will recover in a warmedcage with water and food easily accessible. Gentamicin (5 mg/kg,intramuscular) will be administered immediately post-surgery and thendaily for seven days. The analgesic, Buprenex (0.01 mg/kg of 0.3 mg/mL,subcutaneous;) will be delivered post-surgery and daily for 2 days tominimize animal discomfort. The rats were maintained for 1 week or 9weeks after injury. For each time point and severity of injury, 20animals will be treated and 10 animals will serve as controls. Harvestedtissues will be examined for cavitation, gliosis and axonalregeneration.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theInvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An isolated polypeptide comprising the sequenceaccording to general formula IZ1-X1-X2-X3-X4X5-X6-X7-X8-X9-X10-Z2  (Formula I) (SEQ ID NO: 69) whereinZ1 and Z2 are independently absent or are transduction domains; andwherein X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 is an amino acid sequenceselected from the group consisting of: the amino acid sequenceconsisting of KALARQLGVAA (SEQ ID NO: 62) and the amino acid sequenceconsisting of KALNRQLAVAA (SEQ ID NO: 66).
 2. A composition comprisingone or more polypeptides of claim 1 and a pharmaceutically acceptablecarrier.
 3. A biomedical device comprising one or more isolatedpolypeptides according to claim 1, wherein the one or more isolatedpolypeptides are disposed on or in the device.
 4. The biomedical deviceaccording to claim 3, wherein the one or more isolated polypeptides aredisposed in a matrix disposed on the device.
 5. The biomedical deviceaccording to claim 4, wherein the matrix is a heparin coating.
 6. Theisolated polypeptide of claim 1, wherein X1 is KA, X2 is L, X3 is A, X4is R, X5 is Q, X6 is L, X7 is G, X8 is V, X9 is A, and X10 is A (SEQ IDNO: 62).
 7. The isolated polypeptide of claim 6, wherein Z1 is presentand is a transduction domain.
 8. The isolated polypeptide of claim 7,wherein the polypeptide is YARAAARQARAKALARQLGVAA (SEQ ID NO: 36).
 9. Acomposition comprising the isolated polypeptide of claim 8 and apharmaceutically acceptable carrier.
 10. A biomedical device comprisingthe isolated polypeptide according to claim 8, the polypeptide isdisposed on or in the device.
 11. The biomedical device according toclaim 10, wherein the isolated polypeptide is disposed in a matrixdisposed on the device.
 12. The biomedical device according to claim 11,wherein the matrix is a heparin coating.
 13. The isolated polypeptide ofclaim 8, wherein the polypeptide is synthetic.
 14. The isolatedpolypeptide of claim 8, wherein the polypeptide comprises D-amino acids,L-amino acids, or combinations thereof.
 15. The composition of claim 9,wherein the pharmaceutically acceptable carrier is selected from thegroup consisting of water, Ringer's solution, sodium chloride solution,and oil.
 16. The composition of claim 9 in a dosage form selected fromthe group consisting of oral, buccal, parenteral, topical, inhalation,or rectal dosage forms.
 17. The composition of claim 16, wherein thedosage form is a parenteral dosage form and the parenteral dosage formis selected from the group consisting of subcutaneous, intramuscular,intravenous, intrathecal, intrasternal, and infusion dosage forms. 18.The composition of claim 16, wherein the composition is in powder form.19. The composition of claim 17, wherein the composition is injectable.20. The composition of claim 9, wherein the isolated polypeptide is in atherapeutically acceptable amount of about 0.01 μg/kg body weight toabout 10 mg/kg body weight.
 21. The composition of claim 20, wherein theisolated polypeptide is in a therapeutically acceptable amount of about0.05 μg/kg to about 5 mg/kg body weight.
 22. The biomedical device ofclaim 10, wherein the device is selected from the group consisting of apatch, a film, a hydrocolloid, a hydrogel, a foam, a calcium alginate, acellophane, and a biological polymer.
 23. The isolated polypeptide ofclaim 1, wherein X1 is KA, X2 is L, X3 is N, X4 is R, X5 is Q, X6 is L,X7 is A, X8 is V, X9 is A, and X10 is A (SEQ ID NO: 66).
 24. Theisolated polypeptide of claim 23, wherein Z1 is present and is atransduction domain.
 25. The isolated polypeptide of claim 24, whereinZ1 is YARAAARQARA (SEQ ID NO: 26).
 26. A composition comprising theisolated polypeptide of claim 25 and a pharmaceutically acceptablecarrier.
 27. A biomedical device comprising the isolated polypeptideaccording to claim 25, wherein the isolated polypeptide is disposed onor in the device.
 28. The biomedical device according to claim 27,wherein the isolated polypeptide is disposed in a matrix disposed on thedevice.
 29. The biomedical device according to claim 28, wherein thematrix is a heparin coating.
 30. The isolated polypeptide of claim 7,wherein the isolated polypeptide is RQRRKKRGKALARQLGVAA (SEQ ID NO: 38).31. The isolated polypeptide of claim 1, wherein Z1 is YARAAARQARA (SEQID NO: 26).
 32. A composition comprising one or more isolatedpolypeptides of claim 31 and a pharmaceutically acceptable carrier. 33.A biomedical device comprising one or more isolated polypeptidesaccording to claim 31, wherein the one or more isolated polypeptides aredisposed on or in the device.
 34. The biomedical device according toclaim 33, wherein the one or more isolated polypeptides are disposed ina matrix disposed on the device.
 35. The biomedical device according toclaim 34, wherein the matrix is a heparin coating.
 36. The isolatedpolypeptide of claim 31, wherein the polypeptide is synthetic.
 37. Theisolated polypeptide of claim 31, wherein the polypeptide comprisesD-amino acids, L-amino acids, or combinations thereof.
 38. Thecomposition of claim 32, wherein the pharmaceutically acceptable carrieris selected from the group consisting of water, Ringer's solution,sodium chloride solution, and oil.
 39. The composition of claim 32 in adosage form selected from the group consisting of oral, buccal,parenteral, topical, inhalation, or rectal dosage forms.
 40. Thecomposition of claim 39, wherein the dosage form is a parenteral dosageform and the parenteral dosage form is selected from the groupconsisting of subcutaneous, intramuscular, intravenous, intrathecal,intrasternal, and infusion dosage forms.
 41. The composition of claim39, wherein the composition is in powder form.
 42. The composition ofclaim 40, wherein the composition is injectable.
 43. The composition ofclaim 32, wherein the polypeptide is in a therapeutically acceptableamount of about 0.01 μg/kg body weight to about 10 mg/kg body weight.44. The composition of claim 43, wherein the polypeptide is in atherapeutically acceptable amount of about 0.05 μg/kg to about 5 mg/kgbody weight.
 45. The biomedical device of claim 33, wherein the deviceis selected from the group consisting of a patch, a film, ahydrocolloid, a hydrogel, a foam, a calcium alginate, a cellophane, anda biological polymer.